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Noncoding RNAs at H19/IGF2 Locus:
Role in Imprinting, Gene Expression,
and Associated Pathologies
Nahalie Berteaux, Nathalie Spruyt, and Eric Adriaenssens
Contents
1 The H19/IGF2 Locus and the Parental Imprinting Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
1.1 Overview and Description of the 11p15.5 Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
1.2 The Insulator Model of Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
2 The mRNA-Like Noncoding RNA H19 .................................................. 428
2.1 Properties and Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
2.2 Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
2.3 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
3 The Noncoding Antisense RNA 91H ..................................................... 431
3.1 Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
3.2 Hypothesis About 91H Mechanism of Action ...................................... 432
4 H19/IGF2 Locus-Associated Pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
4.1 Hormone-Dependent Cancers (Breast, Uterus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
4.2 Children Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
Abstract Genomic imprinting is a form of epigenetic regulatio n whereby some
genes are silenced according to their parental origin. The H19/IGF2 locus loca ted in
the chromosome 11 in p15.5 is the best characterized imprinted cluster. The locus
generates two types of noncoding RNAs: the mRNA-like noncoding RNA H19 and
the antisense RNA 91H. The regulation of H19 and its closely linked and recipro-
cally imprinted neighbor, IGF2, has been studied intensively both as a model for
understanding imprinting control mechanisms and because of its role in human
diseases. As with all imprinted clusters, H19 imprinted expression is regulated by
an Imprinting Control Region (ICR), which controls interactions between promo-
ters and shared enhancers. The locus functions like an “insulator model” in which
N. Berteaux, N. Spruyt, and E. Adriaenssens (*)
Institut de Biology de Lille, CNRS UMR 8161, 1 rue Pr Calmette, BP 447 59021 Lille Cedex,
France
e-mail: eric.adriaenssens@univ-lille1.fr
V.A. Erdmann and J. Barciszewski (eds.), RNA Technologies and Their Applications,
RNA Technologies, DOI 10.1007/978-3-642-12168-5_19,
#
Springer-Verlag Berlin Heidelberg 2010
419
trans-factors and epigenetic modifications are also required for full expression of
genes. It is well assumed that H19 RNA functions as a riboregulator of which,
expression is developmentally regulated. Elsewhere, the antisense RNA 91H has
been recently discovered as a large and maternally imprinted noncoding RNA. It
plays a role in the paternal IGF2 regulation and is overexpressed in breast cancer.
This original trans-effect may be due to 91H participation in the three-dimensional
organization of the locus, essential for the appropriate expression of genes. In this
chapter, we summarize our current understanding of the molecular and biological
roles of the ncRNAs expressed at the H19/IGF2 domain, both in terms of their
contribution to genomic imprinting cont rol, as well as in terms of cellular targets
they might interact with. We also review knowledge of the locu s-associated patho-
logies such as cancers and children syndromes.
Keywords Non coding RNA Imprinting H19/IGF2 91H Cancer Develop-
mental pathologies
1 The H19/IGF2 Locus and the Parental Imprinting Model
1.1 Overview and Description of the 11p15.5 Locus
The mammalian genome contains a small but growing number of genes that are
subject to genomic imprinting (Edwards and Ferguson-Smith 2007; Verona et al.
2003). Genomic imprinting is a form of epigenetic gene regulation that results in
expression from a single allele in a parent-of-origin-depe ndent manner. This form of
monoallelic expression is essential to normal mammalian development. While the
precise natu re of the initial epigenetic imprint remains an intensively investigated
topic, it is assumed that the parental imprint is set in the germline, when genomes are
in distinct compartments and can be differentially modified. After fertilization, the
parental imprints must survive the reprogramming that takes place in the preimplan-
tation embryo, including DNA demethylation and changes in histones modifications
(Reik et al. 2001). Imprinting is maintained throughout development and then erased
before being reestablished in the next generation’s germline. About 90 genes have
been reported to be imprinted even if some of them probably remain to be discovered
(for a complete list, see http://igc.otago.ac.nz/home.html and http://www.har.mrc.
ac.uk/research/genomicimprinting/maps.html). Despite extensive studies and some
major advancement regarding this intriguing phenomenon, we have not yet fully
characterized the underlying molecular mechanisms of genomic imprinting. Never-
theless, some principal hallmarks of imprinted genes can be listed:
l
Gene expression is allele-specific.
l
Gene expression is often tissue or stage-specific.
l
Many of imprinted genes are found in clusters throughout the genome.
l
The clusters contain two or more imprinted genes over a region that can span
1 Mb or more.
420 N. Berteaux et al.
l
Within each cluster, a common regulating region, which is called “imprinting
control region” (ICR, also called IC for imprinting Center or ICE for imprinting
control element), controls the imprinting of all genes in the cluster and can act
over hundreds of kilobases. ICRs are designed as differentially methylated
regions with parental-specific modifications that determine their activity. Deletions
of this region lead to the loss of imprinting of multiple genes of the cluster
(Leighton et al. 1995; Ripoche et al. 1997).
l
More recently, it has been reported that noncodin g RNA were associated with
imprinted clusters and have an essential role in regulating gene expression.
The H19/IGF2 cluster is located on the human chromosome 11 in p15.5 (homolog
to the murine distal chromosome 7). This 1 Mbp-long domain described in Fig. 1
includes nine imprinted genes and two independent imprinting centers.
M
R
PL23
H19
INS
TH
ASCL2 (MASH2)
PHEMX (TSSC6)
CD81 (TAPA1)
TSSC4
KCNQ1 (KvLQT1)
CDKN1C (p57KIP2)
SLC22A1L (Tssc5, IMPT1)
SLC22A1L-AS (Tssc5-AS
)
TSSC3 (Phlda2)
TRPM5 (MTR1)
NAP1L4
P
M
Telomere
Centromere
IGF-2-A
S
IGF-2
IC2 IC1
91H
KCNQ1DN
Expressed allele
Silent allele
Sense of transcription
IC
Imprinting Center
P
M
Paternal allele
Maternal allele
KCNQ1OT1 (KvLQT1-as , Lit1
)
Fig. 1 Representation of the human chromosomic region 11p15.5. This 1 Mb-locus includes nine
imprinted genes and two imprinting centers. It is delimited by the two maternally expressed genes
TSSC3 et H19 flanked by two nonimprinted genes NAP1L4 et MRPL23 (Tsang et al. 1995; Paulsen
et al. 1998). This region is homologous with the distal region of chromosome 7 in mouse. In spite
of the phylogenetic maintenance if the region over species, it exhibits some structural and
functional discrepancies. Indeed, the TSSC4 gene is imprinted in mouse but not in human (Paulsen
et al. 2000). The TRPM5 gene is imprinted in human but not in mouse (Prawitt et al. 2000).
A TSSC5 antisense transcript has been revealed in human but not in mouse and its imprinted status
has not yet been defined (Crider-Miller et al. 1997; Cooper et al. 1998). The CD81 gene is
imprinted in human and the imprinted status of the INS (Insulin gene) gene is not determined
(Maher and Reik 2000). The ASCL2 gene is imprinted with maternal expression in mouse whereas
its expression is biallelic in human (Miyamoto et al. 2002; Westerman et al. 2001). Usual names
are indicated and followed by secondary names in brackets. Name of antisense transcripts are
underlined and arrows indicating the sense of transcription are depicted below the chromosome
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 421
Numerous imprinted genes are associated to long antisense RNAs that overlap
several genes. The best described examples are Igf-2r/Air, Ube3A/Ube3A-as, and
Kcnq1/Kcnq1ot1 (Fig. 2). The first demonstration of a direct implication of an
MKRN3
a
b
c
UBE3A
MAGEL2
UBE3A-as
SNRPN ATP10CNDN
AS-SRO
PWS-SRO
ICE
Slc22a1Igf2rMas1 Slc22a2 Slc22a3
Air
Tssc4Ltrpc5
KnDMR1
Cd81 Tssc6 Ascl2Tssc3 Tssc5 Cdkn1c
Kνlqt1-as, Lit1
Kνlqt1
Fig. 2 Antisense RNA associated to imprinted clusters. Igf-2r/Air, Ube3A/Ube3A-as, and Kcnq1/
Kcnq1ot1 are the three best described examples of long antisense RNA, which take part in
epigenetic modifications and gene silencing within imprinted loci. (a) The region of chromosome
15q11–q13 responsible for the Angelman and Prader–Willi syndromes contains a number of
imprinted genes that are coordinately regulated by an imprinting center that contains two func-
tional elements, the PWS-SRO and the AS-SRO. The 460 kb-long Ube3A-as RNA is initiated in
the imprinting center from the paternal allele. It acts as a host gene for the transcription of several
snoRNA (small nucleolar RNA) and represses the UBE3A gene on the paternal allele (Rougeulle
et al. 1998; Runte et al. 2001; Landers et al. 2004). (b) The IGF2R locus. The imprinting center
(ICE) produces a paternally expressed 108-kb long transcript called Air that is necessary for the
silencing in cis of the genes IGF2R, Slc22a1, Slc22a2, and Slc22a3. Air could be implicated in the
methylation spreading thought the locus. (Rougeulle and Heard 2002; Sleutels et al. 2002).
(c) Within the 11p15.5 region, the Kcnq1 gene contains within its intron 10 the imprinting center
KvDMR1, which harbors bidirectional silencing property. This is linked to a paternal antisense
RNA, Kcnq1ot1 (also called Lit1) initiated from KvDMR1. The Kcnq1ot1 promoter shows a
maternal specific methylation. Kcnq1ot1 transcript has a key role in silencing of genes contained in
the Kcnq1 gene imprinted region and it participates to both silencing activity and methylation
spreading (Pandey et al. 2004; Thakur et al. 2004)
422 N. Berteaux et al.
antisense RNA concerns the Air transcript. It consists of a 108 kb-long transcript
localized in the imprinting center within the IG F2r gene and is necessary for the
paternal repress ion of the gene of the locus (Rougeulle and Heard 2002; Sleutels
et al. 2002). In spite of its well-established role in imprinting process, the molecular
mechanism remains unclear and authors propose hypothesis of methylation propa-
gation from the IGF2R gene or of repressive ARN/protein complexes formation.
The 460 kb-long Ube3A-as RNA is initiated in the imprinting center of the
Prader–Willi syndrome. It acts as a host gene for the transcription of several
snoRNA (small nucleolar RNA) and represses the UBE3A gene on the paternal
allele (Rougeulle et al. 1998; Runte et al. 2001; Landers et al. 2004).
Within the 11p 15.5 regi on, the ICR2 i s located within the intron 10 of the
Kcnq1 gene and harbors bidirectional silencing property. This feature is linked to
an antisense RNA, Kcnq1ot1 (also called Lit1 ), of w h ich the prom oter is
containedinICR2.Expressionofthistranscript is exclusively paternal. Indeed,
the Kcnq1ot1 promoter shows a maternal-specific methylation. This differential
epigenetic mark is lost in patients affected by Beckwith–Wiedemann syndrome
(BWS) with RNA biallelic expressi on (Mitsuya et al. 1999; Lee et al. 1999;
Du et al. 2004). More recently, Pandey et al. (2004) have documented that the
Kcnq1ot transcript has a key role in silencing o f genes contained in the Kcnq1
gene imprinted region and that it participates di rectly or indirectly to the methyl-
ation but without RNA interference mechanisms. Furthermore, interruption of
Kcnq1ot1 RNA pr oduc tion by t he inserti on of a polyade nyl atio n sequenc e down-
stream of the promoter also caused a loss of both silencing activity and methyla-
tion spreading. Thus, the antisense RNA plays a key role in t he silencing function
of the ICR (Thakur et al. 2004).
Elsewhere, a noncoding antisense RNA has also been described in the mouse
and human IGF2 genes (Moore et al. 1997; Okutsu et al. 2000). Like IGF2 , this
2.2 kb mRNA tran script is maternally imprinted and overexpressed in Wilms’
tumors. IG F2 -AS was expressed at levels comparable with IGF2 sense expression
derived from promoters P1 and P2 in normal tissue and in breast, ovarian, and
Wilms’ tumor tissues. It is composed of three exons, which overlap the exons 3 and
4 of the IGF2 gene (Vu et al. 2003). Its function remains unknown and its
involvement in imprinting has not yet been demonstrated, but findings indicate
that it is a good marker of Wilms’ tumor (Okutsu et al. 2000).
Finally, discovery of microRNAs (miRNA) and RNA interference could likely
provide new insights on imprinting mechanism. Then, the group of Cavaille has
identified in mouse a short cluster of maternally expressed miRNA genes (miR-431,
miR-433, miR-127, miR-434, and miR-136) transcribed and processed from a gene
antisense to the paternally expressed Rtl1 gene (Retrotransposon-like gene1) (Seitz
et al. 2003). Rtl1, also called Peg11 in sheep, displays homology with the Ty3/gypsy
retrotransposon family and its function is currently unknown (Charlier et al. 2001;
Youngson et al. 2005). Due to this peculiar sense–antisense organization, the
encoded miRNAs are obviously perfectly complementary to Rtl1 mRNA and thus
were predicted to cleave Rtl1 mRNA via RNAi-like mechanisms (Seitz et al. 2003).
Indeed, the predicted RNAi-mediated cleavage sites in the middle of the RNA
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 423
duplexes have been experimentally mapped by 5
0
-RACE (rapid amplification of
cDNA ends) experiments (Davis et al. 2005). These imprinted miRNAs are there-
fore among the very rar e miRNAs in animals that act as siRNAs (small interfering
RNAs) (Yekta et al. 2004). From an evolutionary point of view, this trans-allelic
RNAi between imprinted genes (for example, maternally expressed miRNAs
silence a paternally expressed gene) can be viewed as a good illustration of the
parent-conflict theory (Wilk ins and Haig 2003; for further discussion, see also
Davis et al. 2005; Lewis and Redrup 2005). In addition, this regulation might
also account for the complex gene regulatory network occurring at the ovine
Dlk1–Gtl2 locus, the so-called polar overdominance phenomena (for further infor-
mation, see Davis et al. 2005; also reviewed in Royo and Cavaille
´
2008).
Interestingly, a 23-nucleotide microRNA miR-675 was shown recently to be
processed from the H19 gene and this may in turn regulate mRNAs in development
and/or in oncogenesis. It is endogenously expressed in human keratinocytes and
neonatal mice and overexpressed in cells transfected with human or mouse H19
expression plasmids. These data demonstrate that H19 can function as a primary
microRNA precursor (Cai and Cullen 2007).
1.2 The Ins ulator Model of Imprinting
The H19 gene is one of the first genes proven to be imprinted. In human, it lies
within 200 kbp downstream of the IGF-2 gene (Zemel et al. 1992). The regulation
of H19 and its closely linked and reciprocally imprinted neighbor, IGF2, has been
studied intensively both as a model for understanding imprinting control mechanisms
and because of its role in human diseases. The two genes are imprinted in an opposite
manner, with the paternal IGF-2 and the maternal H19 alleles being reciprocally
expressed (Giannoukakis et al. 1993; Zhang and Tycko 1992).
1.2.1 DNA Methylation of H19 and IGF2 Genes
The H19 silent paternal allele exhibits several characteristics associated to its
transcriptional repression: it is hypermethylated in the promoter region and in the
5
0
region in embryonic tissue s, the promoter shows a compact chromatin structure
(Bartolomei et al. 1993; Ferguson-Smith et al. 1993) and its histone acetylation rate
is lower than the one of the maternal allele (Grandjean et al. 2001).
Surprisingly, the IGF2 promoter region is not methylated and its chromatin
structure is favorable to a biallelic transcription (Sasaki et al. 1992). However,
two other regions preferentially methylated on the expressed paternal allele have
been identified within the gene: the DMR1 located 3 kbp upstream the P1 promoter
acts as a silencer on the maternal allele when it is unmethylated, and the DMR2,
located within exons 5 and 6 is an activator on the paternal allele when it is
methylated (Feil et al. 1994; Murrell et al. 2001; Constancia et al. 2000). It is
424 N. Berteaux et al.
interesting to notice that both regions acquire this differential methylation after
fecundation (it is then a secondary methylation by opposition to primary methylation
that it is established in gamete and allows to distinguish the two parental alleles), and
this implies that it is not responsible for imprinting establishment.
1.2.2 Histone Modifications at the H19/IGF2 Locus
Analysis of histone acetylation state at the murine locus shows that H4 histone
acetylation discrepancies take place within H19 and IGF2 genes in a parental-
specific manner with expressed alleles being more acetylated than silent alleles.
However, the link between DNA methylat ion, histone hypoacetylation, and gene
expression is established only for the H19 promoter region (Grandjean et al. 2001;
Pedone et al. 1999). Moreover, several studies show that the inhi bition of histone
deacetylases deregulates gene expression at the locus with a repression of the H19
active maternal allele, changes in acetylation patterns of the ICR region (Grandjean
et al. 2001), and an IGF2 biallelic expression (Hu et al. 1998; Yang et al. 2003).
Finally, the HDAC recruitment is directly involved in the repression effect of the
insulator protein CTCF described in the next paragraph (Lutz et al. 2000).
1.2.3 The ICR or Imprinting Control Region
The methylation of promoter regions is not sufficient to explain the reciprocal
expression of the two genes, other elements exhibiting primary methylation hall-
marks have been searched within the locus. Discovery of the ICR, located between
the H19 and IGF2 genes, allows understanding the mechanism of imprinting setting.
Existence of a common regulatory region has been initially suggested by dele-
tions assays of the H19 gene region (Leighton et al. 1995; Ripoche et al. 1997). This
sequence, named ICR, is located within the region comprised between 2 and 4 kb
upstream of the transcription start site of the H19 gene and carries primary
methylation marks (Tremblay et al. 1997). ICR has a long-range action to establish
the H19 and IGF2 imprinting during the embryonic development. It is exclusively
methylated on the paterna l allele and shows nuclease hypersensibility sites on the
maternal allele (Hark and Tilghman 1998; Kanduri et al. 2000a). These epigenetic
hallmarks are a sign of a protein binding to the sequences. ICR deletion leads to loss
of H19 and IGF2 (Thorvaldsen et al. 1998, 2002). ICR is also necessary to the
imprinting of a H19 transgene in mouse (Elson and Bartolomei 1997). These studies
show the pivotal role of ICR in imprint ing.
To understand the long-range and allele specific effect of ICR, deletion and
relocalization assays have been conducted. It revealed the chromatin insulator
activity of the ICR region (Webber et al. 1998; Kaffer et al. 2000; Kanduri et al.
2000b). Indeed, it is able to insulate communication between a promoter and
an enhancer when it is located between the two regions. This activity required a
zinc-finger protein named CTCF (CCTC-binding factor) (Bell et al. 1999). In vitro
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 425
and in v ivo experiments have shown in mouse that CTCF binds unmethylated ICR
via four consensus sites and the binding is abolished by DNA methylation (Bell and
Felsenfeld 2000; Hark et al. 2000; Kanduri et al. 2000b; Szabo et al. 2000;
Holmgren et al. 2001; Ulaner et al. 2003). In human, there are seven binding sites
but the sixth is only the one to possess a differential methylation (Takai et al. 2001).
The H19/IGF2 insulation model is represented in the Fig. 3. Both genes share a
common set of enhancers located downstream from the H19 gene. In the mater nal
allele, the CTCF recruitment on the unmethylated ICR acts as a chromatin bound-
ary and blocks the enhancer access to the IGF2 promoter to prevent its activation.
The H19 gene is then activated (Reik and Murrell 2000; Wolffe 2000).
H19
IGF-2
×
EnhICRHucDMR1
M
P
H19
×
++
+
+
INSULATOR
–2–12–137 +10.5 kpb
CTCF
CTCF
INSULATOR
DMR2
+
IGF-2
CTCF
Fig. 3 Reciprocal imprinting mechanism of H19 and IGF2 genes. Activation of gene expression
is indicated by (þ), repression by (), and inhibition of the enhancer function is represented by a
vertical bar. Relative positions are expressed in kilobase pairs relatively to the H19 transcription
start site. Three principal mechanisms intervene in the regulation: the methylation (represented by
vertical bars), the enhancer activity, and the insulator activity (Hark et al. 2000). Three DNA
regions are differentially methylated according to the allele, the DMR1 and 2 of the IGF2 gene
(violet diamond) and the ICR (blue oval) at 2 kb upstream of the H19 gene. The enhancer
sequences Enh (Enhancer downstream of the H19 gene, green circles) and Huc (enhancers
upstream of the H19 gene, blue circles) represent, respectively, the endodermic and mesodermic
enhancers (Ishihara et al. 2000; Drewell et al. 2002). On the maternal allele, the nonmethylated
ICR contains four consensus CTCF binding sites (Hark et al. 2000). The CTCF DNA binding
produces then a chromatin boundary, which prohibits enhancers to access to the IGF2 gene. The
Huc enhancers may also activate the H19 gene (Drewell et al. 2002). The nonmethylated IGF2
DMR1 (violet diamond ) acts as a silencer (Constancia et al. 2000). On the paternal chromosome,
the methylated ICR does not bind any protein but acts as a H19 expression repressor. The Enh
enhancers can then activate the IGF2 promoter and the methylated IGF2 DMR2 also activates
gene expression (Murrell et al. 2001). ICR has a role of transcriptional repressor for the H19 gene
(Srivastava et al. 2000). In 3
0
of H19, a secondary chromatin boundary independent of the
methylation delimits the imprinted domain (Ishihara and Sasaki 2002)
426 N. Berteaux et al.
On the paternal allele, the ICR methylation does not allow CTCF binding and
leads to activation of the distal IGF2 promoter and gene expression (for a review,
see Lewis and Murrel 2004 ). Finally, the maintenance of the unmethylated state of
maternal ICR is due to the CTCF protein, which prevents the de novo methylation
in this region (Fedoriw et al. 2004; Lewis and Murrel 2004; Szabo et al. 2004). The
mesodermic enhancer activity recently discovered (Drewell et al. 2002), which
intervene in the regulation can be added to this model. Finally, the in vivo CTCF
binding upstream of the Mrpl23 gene could be a chromatin boundary delimiting the
imprinted domain (Ishihara and Sasaki 2002).
1.2.4 Imprinting and Parental Specific Chromatin Loops
The cis elements described previously have a long-range action. They have to
physically interact with each other or with their target to exert their eff ects.
Chromosome conformation capture (3C) analysis in mice, which assay for physical
interactions betwee n chromosomal regions, have suggested that CTCF has a critica l
role in the epigenetic regulation of high-order chromati n structure and gene silenc-
ing over considerable distances in the genome, but the precise nature and function
of the looping is debated (Engel et al. 2008; Kurukuti et al. 2006; Lopes et al. 2003;
Murrell et al. 2004; Yoon et al. 2007). Kurukuti et al. 2006 reported that on the
paternal allele, enhancers interact with the IGF2 promoters whereas on the mater-
nal, this is prevented by CTCF binding within the H19 ICR. They demonstrated that
the maternal-specific silencing of IGF2 results when the ICR interacts with a matrix
attachment region (MAR3) and a differentially methylated region (DMR1) at the
IGF2 locus to generate a tight loop around the IGF2 gene, thereby physically
impeding Igf2 expression. Moreover, CTCF interacts with the three clustered
IGF2 promot ers and recruits polycomb repress ive complexes that lead to the
allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to the suppres-
sion of the maternal IGF2 promoters (Li et al. 2008). Elsewhere, Murrell et al.
(2004) reported that on the maternal allele, the unmethylated ICR binds to the
DMR1 of IGF2 resulting in an inactive domain where IGF2 is away from the
enhancers. On the paternal allele, the methylated ICR associates with the methy-
lated IGF2 DMR2 moving IGF2 into the active chromatin domain (Dekker et al.
2002). More recently, it has been shown that on the maternal allele, the enhancers
make contacts throughout the H19 coding unit and promoter (Engel et al. 2008;
Kato and Sasaki 2005).
Figure 4 provides a simplified overview of available data about chromatin loop
structures at the H19/IGF2 locus (Kurukuti et al. 2006; Murrell et al. 2004; Weber
et al. 2003). Additional mechanisms exist for an imprint mark, such as chromatin
composition, organization, and histone acetylation or methylation state (Fuks 2003;
Grandjean et al. 2001), even if DNA methylation is by far the best candidate
(Bestor 2000).
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 427
2 The mRNA-Like Noncoding RNA H19
H19 encodes a spliced and polyadenylated RNA that lacks conserved open reading
frames (ORFs) but does have a conserved secondary RNA structure (Juan et al.
2000). Even if extensive deletions and/or point mutations of an ectopic human H19
RNA generate a protein, (Joubel et al. 1996) no endogenous transl ation product has
so far been identified (Pachnis et al. 1984, 1988). Therefore, it was quickly proposed
that H19 RNA functions as a riboregulator (Brannan et al. 1990) of which
Maternal allele
Paternal allele
CTCF
H19
DMR2
CTCF
ICR
enhancers
H19
MAR3
IGF2
DMR1
H19
DMR2
ICR
enhancers
MAR3
DMR2
ICR
DMR1
Fig. 4 Chromatin loop structures at the H19/IGF2 locus. Chromosome conformation capture (3C)
assays revealed long-range physical interactions within the H19/IGF2 region. These chromatin
structures are orchestrated by CTCF and regulate epigenetic hallmarks and gene silencing within
the locus. On the maternal allele, CTCF binding to the ICR prevents interaction between enhancers
and IGF2 promoter. ICR interacts with a matrix attachment region MAR3 and with the differen-
tially methylated region DMR1 within the IGF2 region. This leads to the formation of a silencing
loop, which impedes IGF2 expression. Enhancers can interact with the H19 promoter and activate
its expression (Engel et al. 2008; Kato and Sasaki 2005). Moreover, CTCF interacts with the three
clustered IGF2 promoters and recruits polycomb repressive complexes that lead to the allele-
specific histone methylation and to suppression of the maternal IGF2 promoters (Li et al. 2008).
On the paternal allele, enhancers interact with the IGF2 promoters (Kurukuti et al. 2006). The
methylated ICR associates with the methylated IGF2 DMR2 moving IGF2 into the active
chromatin domain (Dekker et al. 2002). ICR methylation spreads to the H19 promoter that impairs
H19 paternal expression
428 N. Berteaux et al.
expression is developmentally regulated. It is abundantly expressed in both extra-
embryonic and fetal tissues and is repressed after bir th except in a few adult organs,
particularly in the mammary gland (Douc-Rasy et al. 1993; Dugimont et al. 1995). It
should be emphasized that the role of the H19 gene in cancer is still a matter of
debate. It has been proposed that H19 functions as a tumor suppressor in some
Wilms’ tumors, embryonic rhabdomyosarcoma, and the Beckwith–Wiedmann cancer
predisposing syndrome (Okamoto et al. 1997; Steenman et al. 1994). Consistently,
some studies conclude that it downregulates the IGF2 factor (Wilkin et al. 2000). By
contrast, other studies including ours argue in favor of an oncogenic role of H19 with
a positive correlation with cell aggressiveness (Lottin et al. 2002a; Rachmilewitz
et al. 1995). H19 activation has also been reported in various cancer tissues:breast
(Douc-Rasy et al. 1993; Dugimont et al. 1995; Adriaenssens et al. 1998), bladder
(Ariel et al. 1995; Elkin et al. 1995), lung (Kondo et al. 1995), and esophageal
cancers (Hibi et al. 1996). Its oncoge nic role has been well documented in the
bladder, since it is considered as an oncodevelopmental marker (Cooper et al. 1996)
and regulates genes involved in metastasis and blood vessel development (Ayesh
et al. 2002). These observations support a H19 role in tumor invasion and angiogen-
esis. In spite of polemic on the H19 role in cancer, we clearly established the
oncogenic role of H19 (Lottin et al. 2002a) in breast cancer, and we demonstrated
that H19 overexpression in breast cancer cells promotes the cell cycle progression,
by increasing S phase entry (Berteaux et al. 2005).
2.1 Properties and Expression
The H19 gene was discovered in the mouse as a gene under coordinate regulation
with (-feto-protein in the liver (Pachnis et al. 1984). More recently, Juan et al.
(2000) brought evidence for evolutionarily conserved secondary structure in the
H19 RNA from several mammalian species. The H19 gene is an unusual gene, in
that it is transcribed by RNA polymerase II, processed by capping, splicing, and
polyadenylation; but it does not appear to encode a protein. Actually, the particu-
larity of the H19 transcript is its inability to be translated when the 5
0
untranslated-
region (5
0
UTR) is not experimentally altered (deletions and/or point mutations)
(Pachnis et al. 1988; Joubel et al. 1996). Furthermore, hypothetical translation of
established sequences from a range of mammalian species shows an absence
of conserved ORFs of any size. Consequently, given the evolutionary conservation
of structure at the RNA level and the absence of conservation at the protein level, it
has been proposed that the mature transcript is the functional product of the H19
gene and that its function requires the ability to fold into a specific secondary
structure. As early as 1990, Brannan et al. (1990) proposed that this gene could act
as a “riboregul ator .”
The H19 gene encodes one of the most abundant RNAs in the devel oping mouse
and human embryo (Pachnis et al. 1984; Brannan et al. 1990). It is expressed at the
blastocyst stage of development and accumulates to high levels in tissues of
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 429
endodermal and mesodermal origins (Poirier et al. 1991; Lustig et al. 1994) as well
as ectodermal origin (Ohlsson et al. 1994, Hemberger et al. 1998). H19 is expressed
in the choroid plexus and leptomeninges of the developing mouse fetus (Svensson
et al. 1995) but not in these tissues during human development (Ohlsson et al.
1994). After birth, the gene is repressed in almost all tissues except skeletal muscle
(Pachnis et al. 1984; Leibovitch et al. 1995; Douc-Rasy et al. 1993; Milligan et al.
2000). In other respects, a basal but significant H19 expression is detected at
adulthood in lung, heart, and thymus (Poirier et al. 1991), mammary gland
(Douc-Rasy et al. 1993; Dugimont et al. 1995; Adriaenssens et al. 1999), adrenal
gland (Liu et al. 1995), and uterus (Adriaenssens et al. 1999; Ariel et al. 1997).
2.2 Functions
Since the first mention of H19 in 1984 by Pachnis et al., its functions have only
begun to emerge. It has been reported that H19 RNA was involved in the repression
of the IGF2 oncogene by affecting its transcription (Wilkin et al. 2000) or its
translation (Li et al. 1998). In addition, we brought evidence that the H19 gene
posttranscriptionally upregulates the thioredoxin level, a key protein of the cellular
redox metabolism (Lottin et al. 2002b). Deletion of the H19 gene in mouse
(KO mice) leads to a size and weight increase of about 10% (Ripoche et al.
1997). But the consecutive biallelic IGF2 expression in these animals does not
allow concluding to an H19 direct effect. However, the group of Surani demon-
strated that induction of a targeted H19 biallelic expression via specific silencer
deletion in transge nic mice leads to smaller animals whereas IGF2 expression is not
affected (Drewell et al. 2000). So, the H19 expression pattern suggests that the
transcript assures a major functi on during the development.
2.3 Regulation
Beyond epigenetic regulations, the H19 gene is submitted to local regulations (i.e.,
on the level of the promoter), by hormones, growth factors, or other cytokines.
Indeed, in mammary cells, H19 is activated by HGF-SF (hepatocyte growth factor/
scatter factor), which has been identified as one of the main paracrin mediators of
morphogenetic epithelial/mesenchymal interactions. This factor has potent moto-
genic, mitogenic, and morphogenic effects on epithelial cells in culture, and H19
RNA synthesis is related to the migratory phenotype of cultured cells. EGF and
FGF-2 also activates H19 but less, whereas IGF-2, TGFb1, and TNFa have no
effect on H19 expression in these cells (Adriaenssens et al. 2002). In uterus, during
estrus (proliferative phase) and metestrus (early secretory phase) phases, and in
breast, during puberty and pregnancy, morphologic changes are associated to peak
of H19 expression. Moreover, its expression is regulated by steroid hormones
430 N. Berteaux et al.
estrogen and progesterone, which respectively up- and downregulate the gene
(Adriaenssens et al. 1999). Elsewhere, we also demonstrated a negativ e regulation
of H19 by the tumor suppressor protein P53 (Dugimont et al. 1998).
H19 and AFP (a-fetoprotein) are abundantly transcripted in mammalian fetal
liver but are rapidly repressed after birth. This repression is partly controlled by the
Afr1 locus (a Fetoprotein Regulator 1) (Pachnis et al. 1984). Recently, Perincheri
et al. (2005) have mapped the locus and identified the murine Zhx gene (Zinc
Fingers and Homeoboxes gene), ortholog to the human ZHX2 gene. This factor is
directly responsible for the H19 postnatal repression in liver but also probably in
other organs since it is expressed in ubiquist manner.
In breast cancer, preferential accumulation of H19 transcripts has been observed
at stroma/ epithelium interface (Dugimont et al. 1995; Adriaenssens et al. 1998).
Moreover, scattering and morphogenesis of epithelial cells by a conditioned
medium from fibroblasts induces activation of the H19 gene (Adriaenssens et al.
2002). The H19 gene is then finely regulated by environmental factors and it is
involved in the epithelial/mesenchymal crosstalk, essential during tumorigenesis.
Posttranscriptional regulations have been also reported such as RNA stabilization
by not yet identified proteins (Milligan et al. 2000; Jouvenot et al. 1999) Elsewhere,
a direct association of the human and mouse H19 RNA with the IMP protei n
family have been demonstrated (In mouse: CRD-BP (cMyc mRNA coding Region
instability Determinant Binding Protein) and in human: IMP1, 2, and 3 (IGF-II
mRNA-binding protein)). These proteins are able to regulate H19 RNA localization
and stabilization and are also able to bind the IGF2 RNA (Tessier et al. 2004; Runge
et al. 2000; Nielsen et al. 2001, 2004; Liao et al. 2005).
3 The Noncoding Antisense RNA 91H
3.1 Characterization
A more recently identified characteristic of imprinted genes is their association, in
some cases, with noncoding antisense transcripts (ncRNAs), which have been
suggested to constitute a new epigenetic regulatory system (see Fig. 2). These
ncRNAs are not yet clearly classified, but categories with known gene regulatory
functions are emerging. It includes (1) intergenic transcripts that regulate local
chromatin activity, (2) cis-acting long ncRNAs such as Xist involved in chromo-
some inactivation, and (3) ncRNA expressed within imprinting loci such as Air,
KCNQ1OT1, and UBE3A-as involved in domain silenc ing (Rougeulle and Heard
2002; Rougeulle et al. 1998; Sleutels et al. 2002; Thakur et al. 2004).
These latter transcripts share some characteristics:
l
It is always very long RNA (several hundreds of base pairs).
l
Its expression is allele-specific.
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 431
l
The promoter is often located near an imprinting center and/or in an intron of a
coding gene.
l
The transcript is implicated in gene silencing and epigenetic modifications.
It regulates expression of overlapped (or not) genes in cis.
Apart from the identification of some IGF2 antisenses transcripts, few data are
available on that topic at the H19/IGF2 locus. We recently identified and character-
ized a new transcript within this locus. It consists of a large intergenic 120 kb-long
RNA that we named 91H since it is transcribed antisense to H19. This nuclear and
short-lived RNA is expressed predominantly from the maternal allele in both mouse
and human within the H19 gene region. Moreover, the transcript is stabilized in
breast cancer cells and overexpressed in human breast tumors. Knockdown experi-
ments showed that 91H inhibition downregulates IGF2 expression in trans. Thus,
91H shares the same characteristics as the other similar ncRNAs do, which are
described above, apar t from its trans effect on IGF2 expression. Indee d, we
demonstrated that the maternal 91H transcript is involved in the maintenance of
the paternal IGF2 gene expression (Berteaux et al. 2008).
3.2 Hypothesis About 91H Mechanism of Action
To unravel this possible trans-effect of the 91H RNA, we envisaged a physical
proximity between homologous chromosomes. Recent works using chromosome
conformation capture technology strongly support the notion of epigenetic chro-
mosomal networks. Several different chromosomes converged on the H19 ICR
simultaneously (Ling et al. 2006; Zhao et al. 2006) probably through the CTCF
protein, and these long-range allele-specific chromosomal associations were linked
with epigenetic regulation of transcriptio n in trans. These data support the possi-
bility that the 91H RNA collaborates in vivo with the establishment of chromo-
somal complexes and that this collaboration leads to activation of the IGF2 paternal
allele. The fact that 91H knockdown only slightly affects gene expression from the
maternal allele seems to indicate that 91H effect is not mediated through the ICR/
CTCF complex. However, other candidates such as enhancer DNA regions may be
considered for interacting with the antisense RNA. Indeed, 91H encompasses a
region including the biallelically transcribed HUC sequences. It consists of regu-
latory elements located just upstream of the ICR, whi ch act as strong mesodermal
enhancers and are supposed to activate IGF2 expression on the paternal allele
(Drewell et al. 2002). Accordingly, we proposed a model to explain the 91H
trans-effect on IGF2 expression in which cooperation between mesodermic
(HUC) and endodermic (ENH) enhancer sequences would be required for full
expression of the gene (Fig. 5). Moreover, 91H may attract repressive chromatin
modifications to the maternal allele by trapping factors responsible for DNA or
histone modifications, which become available for the paternal allele when 91H is
disrupted, resulting in IGF2 expression decrease (Rinn et al. 2007; Yu et al. 2008).
432 N. Berteaux et al.
4 H19/IGF2 Locus-Associated Pathologies
4.1 Hormone-Dependent Cancers (Breast, Uterus)
It should be emphasized that the role of the H19 gene in cancer is still a matter
of debate. Investigations in tumor development are delicate on account of the H19
noncoding state, the lack of knowledge of its mechanism of action, and its
imprinted status. Gene expression has been studied in numerous cancer types, but
the gene status appears to be contradictory since it can be either tumor suppressor or
oncogene depending on the studied model. Table 1 provides a nonexhaustive
general survey of bibliographic data available and illus trates their heterogeneity.
Indeed, it has been proposed that H19 functions as a tumor suppressor in some
Wilms’ tumors, embryonic rhabdomyosarcoma, and the Beckwith–Wiedmann can-
cer predisposing syndrome (Okamoto et al. 1997; Steenman et al. 1 994). Consis-
tently, some studies conclude that it downregulates the IGF2 factor (Wilkin et al.
2000). By contrast, other studies including ours argue in favor of an oncogenic role
of H19 with a positive correlation with cell aggressiveness (Lottin et al. 2002a;
Rachmilewitz et al. 1995). H19 activation has also been reported in various cancer
kbp–2–12–137 +10.5
H19
IGF-2
´
CTCF
MrpL23
H19
IGF-2
Limiting
regulatory
factors
?
91H RNA
ICR
HUC
ENH
ICR
HUC ENH
MrpL23
´
Fig. 5 Model for 91H trans-effect on IGF2 expression. Two sets of enhancers (HUC and ENH
sequences that correspond, respectively, to mesodermic and endodermic enhancers) regulate IGF2
expression. Both would be required for full expression of the gene. In addition, the two IGF2
alleles would be competing for a common limited stock of regulatory elements (methylation/
acetylation/transcription?). On the maternal allele, 91H would block the locus and prevent the
HUC sequences from interaction with any regulatory factors. These latter would be then directed
on the paternal allele, in the HUC region and/or in the IGF2 promoter region, and would cooperate
with the cis endodermic enhancers resulting in IGF2 enhanced expression. (Arrows indicates
positive regulations whereas lines with bars correspond to inhibitions; ICR: Imprinting Control
Region, CTCF: CCTC-binding factor)
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 433
tissues: brea st (Douc-Rasy et al. 1993; Dugimont et al. 1995; Adriaenssens et al.
1998), bladder (Ariel et al. 1995; Elkin et al. 1995), lung (Kondo et al. 1995), and
esophageal cancers (Hibi et al. 1996). Its oncogenic role has been well documented
in the bladder, since it is considered as an oncodevelopmental marker (Cooper et al.
1996) and regulates genes involved in metastasis and blood vessel development
(Ayesh et al. 2002). These observations suppor t a H19 role in tumor invasion and
angiogenesis. In spite of polemic on the H19 role in cancer, the oncogenic role of
H19 has been clearly established in some models. In blad der and uterus cancers,
H19 is directly associated to tumor progression and is considered as tumor marker
(Ariel et al. 2000b; Lottin et al. 2005). Some authors consider H19 as an oncofetal
RNA (Ariel et al. 1997, 2000a). Ohana et al. (2002) proposed an approach of gene
therapy based on the use of H19 regulatory sequences driving the expression of the
Table 1 Overview of bibliographic data about oncogene or tumor suppressor status of the H19
gene
Oncogene Tumor suppressor
Organ, model or
type of cancer
Bibliographic references Organ, model or type
of cancer
Bibliographic references
Transgenic mice Leighton et al. (1995),
Ripoche et al. (1997)
Transgenic mice Drewell et al. (2000)
Bladder Ariel et al. (1995, 1997,
2000b), Elkin et al.
(1995), Cooper et al.
(1996), Ayesh et al.
(2002), Ohana et al.
(2002)
SHE cells Wiseman et al. (1991),
Isfort et al. (1997)
Chorion (JEG-3
cells)
Rachmilewitz et al. (1995) G401 cells (WT) Hao et al. (1993)
Breast Douc-Rasy et al. (1993),
Dugimont et al.
(1995), Adriaenssens
et al. (1998), Lottin
et al. (2002a, b)
JEG-3 cells (Chorion) Li et al. (1998)
Lung Kondo et al. (1995) Children liver,
Hepatoblastomas
Li et al. (1998), Wilkin
et al. (2000)
Uterus Tanos et al. (2004), Lottin
et al. (2005)
Wilms’ tumors
(kidney)
Steenman et al. (1994),
Casola et al. (1997)
Esophagus/colon Hibi et al. (1996), Cui
et al. (2002)
Children muscle,
rhabdomyosarcoma
Casola et al. (1997)
Liver Manoharan et al. (2004) Beckwith–Wiedemann
syndrome
Reik et al. (1995)
Ovary Tanos et al. (1999),
Chen et al. (2000)
Adrenals Liu et al. (1995),
Wilkin et al. (2000)
Head/neck Rainho et al. (2001)
Pharynx Ng et al. (2003)
Testicles Verkerk et al. (1997),
Ariel et al. (2000a)
Demonstration of the oncogene/tumor suppressor status in mentioned works is not always clearly
established. Numerous results converge to one or the other of these statuses and from these data,
authors have proposed their hypothesis
434 N. Berteaux et al.
diphtheria toxin. Injection of these constructs in mice-induced tumors derived from
cancerous colon and bladder cells leads to a decrease of the tumor growth (Ohana
et al. 2005). In breast cancer, H19 is overexpressed in 70% of the adenocarcinomas
and its expression is correlated with the “Tumor values,” the pres ence of hormones
receptors, and tumor invasion (Adriaenssens et al. 1998). Overexpression of an
ectopic H19 gene enhances the tumorigenic properties of breast cance r cells (Lottin
et al. 2002a) and promotes the cell cycle progression, by increasing S phase entry
(Berteaux et al. 2005).
In all cases, deregulation of H19 expression results in genetic alterations leading
to a loss of heterozygote (LOH, for example, in the case of uniparental disomy) or
epigenetic alterations leading to a loss of imprinting (LO I by hypermethylation or
hypomethylation of the H19 promoter or the ICR). It is then often difficult to assign
directly the observed phenotypes to the H19 gene only, since it is tightly connected
to the IGF2 gene, and in most of the cases, the expression of both the genes is
modified. To summarize, the H19 contribution in tumorigenesis depends on the
physiological conditions, the considered tissue, and the nature of deregulations.
4.2 Children Syndromes
The demonstration that the H19 transcript is stabilized in breast cancer cells and
overexpressed in human breast tumors led us to propose a link between antisense
transcription and cancer. Furthermore, we know that the deregulation of the H19/
IGF2 locus is associated to several human fetal syndromes such as BWS and
Silver–Russell syndrome (SRS).
BWS is associated with fetal and postnatal overgrowth and is associated with
embryonic tumors such as children kidney tumors named Wilm’s tumors. BWS can
be caused by a range of different defe cts. Several distinct genetics or epigenetics
errors involving 11p15 have been identified in different BWS patients. Some
patients have maternal chromosomal rearrangements of 11p15, meaning that
there is a disruption of the chromosome in this region. Other patients have paternal
uniparental disomy of 11p15, meanin g that the maternal copy of this region is
replaced with an extra paternal copy. Many other patients have abnormal DNA
methylation in different DMR regions of 11p15, meaning that normal epigenetic
marks that regulate imprinted genes in this region are alter ed. In some cases, the
expression of IGF2 gene is doubled and expression of H19 gene is silenced. BWS is
often associated with H19 epigenetic inactivation due to ICR hypermethylation.
This overexpression of IGF2 is responsible for symptoms linked to the pathology.
Nevertheless, in some cases, the specific defe ct causing BWS in an affected patient
may remain unknown. In about one-third of BWS patients, the genetic or epigenetic
mutation is unknown.
SRS is an intrauterine growth delay associated to an altered postnatal growth with
facial dysmorphy and corporal asymmetry. Reasons of SRS are varied, chromosome
mosaic, equilibrated translocation (1; 17 or 17; 20), deletion (8q11–q13). In some
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression 435
cases, defect in 11p15-5 region has been observed. For numerous patients, this
syndrome is associated with ICR hypomethylation, IGF2 silencing, and H19 biallelic
expression. As BWS, some cases of SRS remain unexplained. The discovery of 91H
gene and its function on gene regulation in 11p15.5 locus can give research lanes to
explore theses unexplained cases of BWS or SRS.
5 Conclusion
There is growing evidence that noncoding transcripts are functional and take part
in most, if not all, complex genetic phenomena in eukaryotes, including RNA
interference-related processes such as transcriptional and posttranscriptional gene
silencing as well as parental imprinting and allelic exclusion. Noncoding RNAs
intervene in general biological processes and the close connection between non-
coding RNAs and epigenet ic processes suggests that they compose a hitherto
hidden layer of genomic programming in humans. The next frontier is now the
functional characterization of these molecules to understand the interactions
between the regulatory RNAs and their targets. This is a way to better understand
how an RNA molecule can regulate cell cycle, gene expression, methylation
spreading, or even chromosomal network. The particular 91H antisense transcript
that is produced at the H19/IGF2 locus adds further complexity to the cluster of
imprinted genes in the human imprinted 11p15.5 region. 91H RNA is involved in
the control of paternal IGF2 expression suggesting that a distinct mechanism of
imprinting arises in the locus. This innovating type of regulation in “trans” between
two homologous chromosomes need to be elucidated to understand, at least in part,
the complex regulation that takes place in the imprinted locus (chromosomal
network, methylation...). Functiona l studies would allow using these RNA mole-
cules as tumor markers or therapeutic targets. Particularly, 91H could be a good
candidate to improve the diagnostic and the therapeutic tools for severe children’s
syndromes or adult cancers associated to the 11p15 chromosomal region.
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