Human imprinted retrogenes exhibit non-canonical imprint chromatin signatures and reside in non-imprinted host genes

Abstract

Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic 'host' gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters.

Full text

Human imprinted retrogenes exhibit non-canonical
imprint chromatin signatures and reside in
non-imprinted host genes
David Monk
1,
*, Philippe Arnaud
2
, Jennifer M. Frost
3,4
, Andrew J. Wood
4
,
Michael Cowley
4
, Alejandro Martin-Trujillo
1
, Amy Guillaumet-Adkins
1
,
Isabel Iglesias Platas
5
, Cristina Camprubi
1
, Deborah Bourc’his
6
, Robert Feil
2
,
Gudrun E. Moore
3
and Rebecca J. Oakey
4
1
Imprinting and Cancer Group, Cancer Epigenetics and Biology Program, Bellvitge Institute for Biomedical
Research (IDIBELL), L’Hospitalet de Llobregat, 08907, Barcelona, Spain,
2
Institute of Molecular Genetics
(IGMM), CNRS UMR5535 and University of Montpellier, 1919 route de Mende, 34293 Montpellier, France,
3
Clinical and Molecular Genetics Unit, Institute of Child Health, University College London,
4
Department of
Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London, SE1 9RT, UK,
5
Servicio de
Neonatologı
´a, Hospital Sant Joan de De
´u, Barcelona, Spain and
6
UMR 3215/Inserm U934, Unite
´de ge
´ne
´tique
et biologie du de
´veloppement, Institut Curie, 26 rue d’Ulm, Paris Cedex 05, France
Received October 7, 2010; Revised November 5, 2010; Accepted November 12, 2010
ABSTRACT
Imprinted retrotransposed genes share a common
genomic organization including a promoter-
associated differentially methylated region (DMR)
and a position within the intron of a multi-exonic
‘host’ gene. In the mouse, at least one transcript
of the host gene is also subject to genomic imprint-
ing. Human retrogene orthologues are imprinted
and we reveal that human host genes are not
imprinted. This coincides with genomic rearrange-
ments that occurred during primate evolution, which
increase the separation between the retrogene
DMRs and the host genes. To address the mech-
anisms governing imprinted retrogene expression,
histone modifications were assayed at the DMRs.
For the mouse retrogenes, the active mark
H3K4me2 was associated with the unmethylated
paternal allele, while the methylated maternal allele
was enriched in repressive marks including
H3K9me3 and H4K20me3. Two human retrogenes
showed monoallelic enrichment of active, but not
of repressive marks suggesting a partial uncoupling
of the relationship between DNA methylation and
repressive histone methylation, possibly due to the
smaller size and lower CpG density of these DMRs.
Finally, we show that the genes immediately
flanking the host genes in mouse and human are
biallelically expressed in a range of tissues, sug-
gesting that these loci are distinct from large
imprinted clusters.
INTRODUCTION
Genomic imprinting is a form of epigenetic gene regula-
tion that results in allelic expression dictated by parental
origin (1). Differential DNA methylation is a major com-
ponent in regulating this process. Discrete differentially
methylated cis-acting regions, known as imprinting
control regions (ICRs), orchestrate the monoallelic expres-
sion of numerous genes within imprinted domains and are
established while the maternal and paternal genomes are
physically separated in their respective germ lines. To
date, all imprinted domains are known to contain
regions of differential DNA methylation (DMRs) that
are deposited in CpG-rich sequences during oogenesis or
spermatogenesis by the DNMT3A/DNMT3L de novo
methyltransferase complex (2–4). A subset of maternally
DNA-methylated germline DMRs require the activity
of the amine oxidase domain 1 containing histone
demethylase AOF1/KDM1. This demethylase is presum-
ably needed to remove any permissive histone H3 lysine 4
(H3K4) methylation present at these CpG islands in the
growing oocytes (5). After fertilization, these regions of
differential DNA methylation are maintained in somatic
*To whom correspondence should be addressed. Tel: +34 93260 7500 (ext. 3165); Fax: +34 93260 7219; Email: dmonk@iconcologia.net
Published online 7 February 2011 Nucleic Acids Research, 2011, Vol. 39, No. 11 4577–4586
doi:10.1093/nar/gkq1230
ßThe Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

tissues by DNMT1 (6), and are associated with numerous
histone modifications.
It has previously been shown that DMRs have a con-
stitutional histone signature that comprises histone H3
lysine 9 trimethylation (H3K9me3), Histone H4 lysine
20 trimethylation (H4K20me3) and symmetrical histone
H2A/H4 arginine 3 dimethylation (H2A/H4R3me2s)
on the DNA methylated allele (7,8). This is in contrast
to the enrichment of the transcriptionally permissive
H3K4me2/3 mark on the unmethylated allele (9). A
number of genes that are imprinted solely in the mouse
placenta, have been shown recently to require allelic re-
pressive histone modifications at their own DNA-
unmethylated promoters to maintain allelic expression
(10–13).
Imprinted genes have diverse evolutionary origins.
Some imprinted genes are products of retrotransposition
from parental genes on the X chromosome. Four im-
printed retrogenes in the mouse—Mcts2,Nap1l5,U2af1-
rs1 (also called Zrsr1) and Inpp5f_v2—are associated with
DMRs at their promoters, and reside in introns of multi-
exonic host genes. In all cases, at least one transcript of the
host is also subject to imprinting. We have previously
shown that the mouse retrogene Mcts2 influences the
choice of polyadenylation (polyA) site for transcripts of
the host gene H13 in an allele-specific manner (14).
Expression of Mcts2 from the paternal allele causes H13
transcripts to terminate upstream of the retrogene. On the
maternal chromosome, H13 utilizes downstream polyA
sites because the Mcts2 DMR is methylated and the
retrogene silenced. A recent transcriptome-wide analysis,
using the ultra sensitive RNA-seq technology which is cap-
able of detecting subtle biases in allelic transcription, has
suggested that one transcript variant of Inpp5f, the host
gene of Inpp5f_v2, and Herc3, the Nap1l5 host gene, are
also subject to isoform-specific allelic expression (15).
To investigate whether the human orthologues of the
X-derived imprinted retrogenes influence allele-specific ex-
pression of their respective host genes, and whether this
influence extends to neighboring genes, we have analyzed
the allelic expression of retrogenes, host and flanking
genes in humans. The U2af1-rs1 gene does not have a
human counterpart. We find that the human orthologues
INPP5F_V2,NAP1L5 and MCTS2 are paternally ex-
pressed in a wide range of fetal tissues, and that their
promoters are embedded in maternally DNA-methylated
regions. In humans, the host genes are not subject to im-
printing, probably due to differing exon/30-UTR positions
in relation to the retrogene integration sites. The genes
immediately flanking the host genes are biallelically ex-
pressed in both mice and humans, showing retrogene-host
pairs do not form parts of larger imprinted clusters. In
mice, the allelic chromatin of these DMRs conforms to
the constitutional histone modification signature with
the repressive modifications H3K9me3, H4K20me3 and
H2A/H4R3me2s enriched on the DNA methylated
allele, and the permissive modification H3K4me2
enriched on the unmethylated allele. These patterns of
histone modifications are not conserved at the human
NAP1L5 and MCTS2 promoters, correlating with
reduced CpG content and CpG island size, which we
speculate may influence the recruitment of the histone
methyltransferases (HMTs).
MATERIALS AND METHODS
Human tissues
A cohort comprising 65 fetal tissue sets (8–18 weeks) with
corresponding maternal blood sample and 96-term placen-
tal samples are from the Moore Tissue bank and is
described elsewhere (16). An additional 96 human
placenta samples were obtained from the Hospital
St Joan De Deu collection (Barcelona, Spain). Normal
peripheral blood was collected from adult volunteers
aged between 19–60-years old. DNA and RNA extraction
and cDNA synthesis were carried out as previously
described (11). Ethical approval for adult blood and
fetal tissue collection was granted by the Hammersmith,
Queen Charlotte’s and Chelsea and Acton Hospital
Research Ethics Committee (Project Registration 2001/
6029 and 2001/6028); Collection of the HSJD placental
cohort was granted by the ethical committee of Hospital
St Joan De Deu Ethics Committee (Study number 35/07).
Cell lines and mouse crosses
Wild-type mouse embryos and placentas were produced
by crossing C57BL/6 females with either Mus musculus
molosinus (JF1) or Mus musculus castaneus (C) male
mice. RNA and DNA from Dnmt3l
/+
mice (BxC,
C57BL/6 mother and Castaneus father) was isolated and
extracted as previously described (2). The E9.5 Dnmt3l
/+
embryos (BxJ) used to assess Nap1l5 expression were a
kind gift from Dr Kenichiro Hata (NRICHD, Okura,
Tokyo, Japan). The human TCL1 and 2 placental tropho-
blast cell lines were grown in DMEM supplemented with
10% FCS and antibiotics.
Allelic expression analysis
Genotypes of DNA were obtained for exonic SNPs
identified in the UCSC browser (NCBI36/hg18,
Assembly 2006) by PCR. Sequences were interrogated
using Sequencher v4.6 (Gene Codes Corporation, MI,
USA) to distinguish informative heterozygote samples.
Informative samples were analysed by RT–PCR in corres-
ponding cDNA using, where possible, intron-crossing
primers that incorporated the heterozygous SNP in the
resulting amplicon (Supplementary Table S1). RT–PCRs
were performed using cycle numbers determined to be
within the exponential phase of the PCR, which varied
for each gene, but was between 32–40 cycles. The RT–
PCRs for HERC3A,HERC3C and both isoforms of
Abcg2 were analyzed by nested RT–PCR, with the first
PCR amplified for 25 cycles, with 5 ml of this product
used as template for the second round PCR which was
limited to 30 cycles.
Real-time qRT–PCR
All PCRs were run in triplicate from the same sample on
either an ABI Prism 7700 sequence detector or a 7900 Fast
real-time PCR machine (Applied Biosystems) following
4578 Nucleic Acids Research, 2011, Vol. 39, No. 11

the manufacturer’s protocol. All primers were optimized
using SYBR Green amplification followed by melt curve
analysis to ensure that amplicons were free of primer
dimer products. Thermal cycling parameters included
Taq polymerase activation at 95C for 10 min for one
cycle, repetitive denaturation at 95C for 15 s, and anneal-
ing at 60C for 1 min for 40 cycles. All resulting triplicate
cycle threshold (C
t
) values had to be within one C
t
of each
other. The quantitative values for each triplicate were
determined as a ratio with the level of Gapdh, measured
in the same sample, with the mean providing relative
expression values.
Analysis of allelic DNA methylation
Approximately 1 mg DNA was subjected to sodium
bisulphite treatment and purified using the EZ GOLD
methylation kit (ZYMO, Orange, CA, USA). Bisulphite
specific primers for each region were used with Hotstar
Taq polymerase (Qiagen, West Sussex, UK) at 45 cycles
and the resulting PCR product cloned into pGEM-T Easy
vector (Promega) for subsequent sequencing. MeDIP was
performed on 6 mg sonicated (Diagenode Bioruptor)
genomic DNA with an average size of 150 bp. Samples
were denatured and incubated with a monoclonal
antibody against 5-methylcytidine (Eurogentec).
Immunoprecipitated DNA was then isolated using IgG
Dynabeads (Dynal Biotech), digested with proteinase K
and phenol–chloroform extraction was followed by
ethanol precipitation. MeDIP enrichment was verified by
duplexed PCR for the methylated SERPIN B5 promoter
and the unmethylated UBE2B promoter. Southern
blotting was performed following standard protocols
using methylation-sensitive restriction enzymes. Digested
DNA was subjected to agarose gel electrophoresis and
transferred to Hybond N+ membrane (Amersham).
Radio-labeled PCR product probes were hybridized
over-night at 65C, and subsequently washed in increasing
stringency SSC/0.1% SDS washes. PCR primers used
to generate probes are listed in Supplementary Table S1.
Chromatin immunoprecipitation
Two adult leukocyte samples and the TCL1 and TCL2 cell
lines were used in addition to E18.5 mouse embryos
for chromatin immunoprecipitation (ChIP). ChIP was
carried out as previously described (11,13) using the fol-
lowing Upstate Biotechnology antisera directed against
H3K4me2 (07-030), H3K9me2 (07-441), H3K9me3
(060904589), H3K9ac (07-352), H3K27me3 (07-449),
H4K20me3 (07-463) (Upstate Biotechnology) and H2A/
H4R3me2s (Abcam ab5823 97454/520317). ChIPed DNA
was subjected to allele-specific PCR. Polymorphisms
within 1 kb of the CpG island were identified by
interrogating SNP databases or genomic sequencing (see
Supplementary Table S1 for primer sequences and
location). Only ChIP sample sets that showed enrichment
for additional ICRs were used in the analysis.
Precipitation levels in the ChIP samples were
determined by real-time PCR amplification, using SYBR
Green PCR kit (Applied Biosystems). Each PCR was run
in triplicate and results are presented as fold enrichment
(comparison to mock) and normalized to the level of pre-
cipitation at the SNURF-ICR, a control for both active
and repressive histone modifications located on human
chromosome 15.
RESULTS
MCTS2 does not influence allelic expression of HM13
The H13 gene on mouse chromosome 2 is known to
generate at least five transcripts, all originating from a
single promoter, but differing in polyA site usage (14).
The utilization of these alternative polyA sites is
influenced by the paternally expressed Mcts2 imprinted
retrogene and the CpG island that comprises its DMR.
The short H13d and etranscripts are paternally expressed,
whereas the H13a,band ctranscripts, that extend through
Mcts2 and the DMR to the canonical polyA site are ma-
ternally expressed (Figure 1A). To assess allelic expression
in humans we identified transcribed SNPs unique to each
isoform, and allele-specific assays were carried out in a
selection of human first trimester fetal tissues and term
placentas. The human MCTS2 gene (also known as
PSIMCT-1, MCTS1-pseudogene) is imprinted in a
variety of fetal tissues (Figure 1B and Supplementary
Table S2). MCTS2 differs from its mouse orthologue as
it can splice into the last eight exons of the HM13 host
gene (Figure 1B). Sequence analyses revealed that in
addition to the highly conserved MCTS2 open reading
frame, this RNA has the potential to be bi-cistronic,
encoding for a chimeric protein lacking the first 151
amino acids of HM13, but sharing 243 amino acids in
the C-terminus.
The MCTS2 promoter is embedded within a DMR
(Figure 1B and Supplementary Figure S1A), while the
HM13 host gene originates from a CpG island that is
unmethylated in all the tissues analysed. Alignment of
human expressed sequence tags (ESTs) revealed a
number of transcripts. The expression of the human
short HM13D isoform (Genbank NM_178982), which ter-
minates prior to the transcriptional start site (TSS) of
MCTS2, and the full-length transcript HM13C
(Genbank NM_030789) are biallelic in all fetal tissues
analysed (Figure 1B and Supplementary Table S2).
The NAP1L5 promoter is not within a CpG island,
but is a DMR
The paternally expressed Nap1l5 retrogene was first
identified in a genome-wide screen for differential DNA
methylation, and was reported to be predominantly pater-
nally expressed in mouse brain (17). The host gene, Herc3,
gives rise to a number of transcript isoforms. At least two
short isoforms (Herc3b and c) are expressed from the
paternal allele in mouse brain [A.J. Wood and R.J.
Oakey, unpublished data, (15)]. The full length Herc3a
transcript has a maternal expression bias, similar to the
full length H13 isoforms (14). We detect paternal expres-
sion of NAP1L5 in all fetal tissues analyzed. The human
HERC3 gene also contains one long (HERC3A, Genbank
NM_014606) and two short isoforms (HERC3B,
Genbank BC038960; HERC3C, Genbank AK296397).
Nucleic Acids Research, 2011, Vol. 39, No. 11 4579

A
B
C
D
E
F
Figure 1. (A) Map of the H13 locus located on mouse chromosome 2, showing the location of the various imprinted transcripts and CpG islands
(red transcripts are maternally expressed, blue are paternally expressed and grey are expressed from both parental alleles. Arrows represent direction
of transcription). (B) Schematic of the human HM13 gene on chromosome 20, showing the distribution of exons and insertion of MCTS2 into intron
4. The methylation status of the HM13 promoter CpG island and MCTS2 CpG island were examined by bisulphite PCR. Each circle represents a
single CpG dinucleotide and the strand. Filled circle, a methylated cytosine; open circle, unmethylated cytosine. The sequence traces show allelic
expression for MCTS2 and HM13 isoforms (for clarity only sequence traces for MCTS2 BC053868 are shown). (C) A map of the Herc3 domain on
mouse chromosome 6. (D) The human NAP1L5 gene and the insertion into intron 22 of HERC3.(E) A schematic map of the Inpp5f gene on mouse
chromosome 7, and (F) the orthologous region on human chromosome 10.
4580 Nucleic Acids Research, 2011, Vol. 39, No. 11

Our allele-specific assays are suggestive of biallelic expres-
sion of all isoforms in all tissues including brain (Figure
1C and D, and Supplementary Table S2), although our
methodology is not directly comparable with RNA-seq,
which can detect subtle expression biases. The HERC3
transcripts initiate from an unmethylated CpG island,
whereas the NAP1L5 promoter is DNA methylated on
the maternal allele, even though the region is not statistic-
ally a CpG island in humans (Figure 1D).
INPP5F_V2 is imprinted in numerous human tissues
We previously identified a neural-specific, paternally ex-
pressed Inpp5f_v2 transcript using expression microarrays
(18). Like the other murine imprinted retrogene loci, the
host gene Inpp5f exhibits isoform-specific expression, with
a maternal expression bias in a truncated shorter tran-
script (Genbank AK039468) (15). In addition, another
transcript, Inppf5_v3, arising from a different promoter,
is paternally expressed in mouse brain (19,20). The
genomic organization of the human locus resembles that
of the mouse, however, there is no evidence for a human
Inpp5f_v3 orthologue (Figure 1E and F). The promoter of
the human INPPF5_V2 transcript is embedded within a
maternally DNA-methylated DMR (Figure 1F and
Supplementary Figure S1B), resulting in paternal expres-
sion in a wide range of tissues (Supplementary Table S2).
The full-length host gene transcripts (Genbank
AB023183), and the truncated isoform (Genbank
BC052367), originate from an unmethylated CpG island
and are biallelically expressed.
Retrogenes-host pairs do not form part of larger
imprinting clusters
In order to determine the boundaries of imprinting at each
retrogene-host locus, we investigated the allele-specific ex-
pression of the genes flanking the host genes in both mice
and humans. We assessed expression in various embryonic
tissues and placenta using allele-specific assays between
crosses of mouse strains C57BL/6 (B) x Mus musculus
castaneus (C). The genes immediately adjacent to Mcts2/
H13,Id1 and Remi, are biallelically expressed in all tissues
at embryonic day E18.5, as are 111007A13RIK and Bag3
flanking Inpp5f_v2/Inpp5f. The Fam13a gene, telomeric to
Nap1l5/Herc3 is expressed from both alleles, while Abcg2,
centromeric to Nap1l5/Herc3, is monoallelically expressed
in the placenta. This reflects a bias in expression from the
C57BL/6 allele and the gene is therefore an expressed
quantitative trait locus (eQTL) and not imprinted
(Figure 2A and Supplementary Figure S2). This conclu-
sion is supported by the persistence of Abcg2 monoallelic
expression in Dnmt3l
/+
placental trophoblast, despite the
loss of imprinting of Nap1l5 (Figure 2B).
A
B
C
Figure 2. (A) A schematic map of the Abcg2 gene, with the location of the alternative promoter regions. The methylation status of the CpG island
associated with isoform 1 was examined in placenta-derived DNA. The allelic expression of Abcg2 is assessed in various fetal tissues in reciprocal
mouse crosses. (B) The allelic expression of Abcg2 and Nap1l5 in placental trophoblasts from Dnmt3l
/+
mice. (C) The allelic expression of the
ABCG2 gene in human term placenta.
Nucleic Acids Research, 2011, Vol. 39, No. 11 4581

To assess the allelic origin of expression for the
orthologous flanking genes in the human, we assessed
the expression of REMI,ID1,BAG3,CORF119 and
FAM13A/FAM13AOS. These genes are expressed
biallelically in all fetal tissues (Supplementary Table S3
and Figure S3). In higher primates, including rhesus
monkey, orangutan, chimps and humans, the organization
of the genes centromeric to NAP1L5/HERC3 is different
to that of mouse and rat, resulting in the gene PIGY
being immediately centromeric to NAP1L5/HERC3, and
ABCG2 300 kb away. In all human fetal tissues
analyzed, including placenta, both PIGY and ABCG2
are biallelic (Figure 2C and Supplementary Figure S3).
These finding strongly suggest that imprinted
retrogene-host pairs do not form part of larger imprinting
clusters.
Histone modification and allelic repression at imprinted
retrogene DMRs
In mouse the imprinted expression of Nap1l5 and
Inpp5f_v2 is restricted to the brain (17–19), whereas in
humans, imprinted expression is observed in a wider
variety of fetal tissues (Figure 1, Supplementary Table
S1 and Figure S4). To date all germline DMRs are
differentially methylated in all somatic tissues, indicating
that DNA methylation on its own is not responsible
for tissues-specific differences in expression observed at
imprinted loci. To investigate whether the discrepancy
in expression profiles we observe between species could
be attributed to histone modifications, we analyzed the
allelic enrichment of both permissive and repressive
histone modifications. Our analysis focused on modifica-
tions on histone H3 and H4, including acetylation of
lysine-9 (H3K9ac) and H3K4me2 as markers of active
chromatin; and the repressive marks of H3K9me3 and
H3K27me3 of histone H3, along with the histone H4
modifications H4K20me3 and H2A/H4R3me2s.
ChIP was performed on native chromatin from brain
and decapitated embryos for both BxC and BxJF1 crosses.
We ascertained allelic enrichment using polymorphisms
mapping within the DMRs of Nap1l5, Inpp5f_v2 and
Mcts2. The active modification H3K4me2 was strongly
enriched specifically on the unmethylated paternal allele
for all three DMRs in both brain and embryo, while
most H3K9ac precipitation was predominantly in brain
(Figure 3), the tissue in which these genes are expressed.
The same regions showed precipitation of the repressive
marks H3K9me3, H4K20me3 and H2A/H4R3me2s on
Mouse ChIP
*
Brain
Input
B6 BxJ JF1
H3K9ac H3K4me2 H3K27me3 H4K20me3
UBUBUBUBUB UB
H3K9me3 H4R3me2s
*
** *
I
npp5f_v2
DMR
Embryo
*
**
mat
pat
mat
pat
Nap1l5
DMR
**
*
*
Input
B6 BxJ JF1
H3K9ac H3K4me2 H3K27me3 H4K20me3
UBUBUBUBUB UB
H3K9me3 H4R3me2s
***
**
*
*
**
Embryo
mat
pat
Brain
mat
pat
Mcts2
DMR
**
Input
B6 BxC Cast
H3K9ac H3K4me2 H3K27me3 H4K20me3
UBUBUBUBUBUB
H3K9me3 H4R3me2s
*
*
*
*
*
** *
*
Embryo
mat
pat
Brain
mat
pat
Figure 3. (A) The allelic precipitation of the mouse retrogene DMRs in embryo and brain tissues. Native ChIP followed by PCR and restriction
digest-mediated allelic discrimination of the input, antibody bound (B) and unbound (U) chromatin fractions on BxJ embryos and brains for Nap1l5
and Inpp5f_v2, and on BxC embryos and brains for Mcts2. The asterisks represent a relative allelic enrichment of >3-fold compared to the unbound
fraction.
4582 Nucleic Acids Research, 2011, Vol. 39, No. 11

the DNA methylated maternal allele (Figure 3). The re-
pressive H3K27me3 mark showed different profiles for
the three mouse DMRs. This modification did not show
allelic enrichment at the Inpp5f_v2 DMR in either brain
or embryo, but was precipitated on the DNA methylated
maternal allele at the Mcts2 DMR. At the Nap1l5 DMR,
we observed that H3K27me3 was precipitated on the
unmethylated paternal allele in embryo but not in brain
(Figure 3). This pattern of enrichment is reminiscent of the
monoallelic bivalent chromatin domain reported at the
Grb10/GRB10 gene (21,22). This monoallelic bivalent
conformation is not detected at the Nap1l5 DMR in
brain, suggesting that the removal of H3K27me3 is con-
comitant with the paternal expression observed for Nap1l5
in mouse brain.
Extensive genotyping of the human NAP1L5,MCTS2
and INPP5F_V2 DMRs revealed that SNPs in these regu-
latory regions are rare. However, we were able to identify
heterozygous samples that allowed us to discriminate
between alleles. The SNP rs2972011 is located 200 bp
from the TSS of NAP1L5, whereas rs7907781 and
rs1115713 are 600 bp and 50 bp from the TSS of
INPP5F_V2 and MCTS2, respectively. To ensure that
these SNPs mapped within the DMRs, we performed
DNA methylation immunoprecipitation (meDIP) using
antisera directed against 5-methylcytosine. This was due
to the difficultly in amplifying bisulphite converted DNA
in the vicinity of SNPs rs7907781 and rs1115713. For all
three regions we observed monoallelic enrichment in
heterozygous placental DNA samples, and where inform-
ative, the DNA methylation was detected on the maternal
allele (Figure 4A). Using these same amplification condi-
tions, we performed ChIP on native chromatin isolated
from adult peripheral blood leukocytes and from two
human placental cell lines, TCL1 and TCL2 (23) for the
NAP1L5 and MCTS2 DMRs. Unfortunately, no hetero-
zygous cell lines could be found that were informative for
INPP5F_V2, despite genotyping of over 140 leukocyte
samples and normal tissue cell lines. Similar to the
mouse, we observe strong monoallelic enrichment for
H3K4me2 at the NAP1L5 and MCTS2 DMRs, but since
no parental DNA samples were available, allelic origin
could not be assigned. Unexpectedly, we did not observe
allelic precipitation for any of the repressive histone marks
at these DMRs, despite strong allelic enrichment at the
SNURF/SNRPN,H19 and MEST DMRs (Figure 4B;
data not shown). To confirm that the histone modifications
were present at the NAP1L5 and MCTS2 DMRs, we per-
formed quantitative ChIP analysis on the placental cell
line TCL1 (Figure 4C). The precipitation values obtained
were normalized to those for the SNURF/SNRPN DMR,
which revealed that H3K4me2 is more abundant at the
MCTS2 and NAP1L5 promoters, whereas, the repressive
histone modifications were precipitated several fold less.
Interrogation of human histone maps [http://dir.nhlbi.nih
.gov/papers/lmi/epigenomes/hgtcell.aspx, and (24)] con-
firmed the absence of significant enrichment for these
repressive marks at 1–2 nucleosomes resolution
A
BC
Figure 4. (A) Methylation-immunoprecipitation was performed on placental DNA using anti 5mC antibody. The efficiency of IP was assessed by
PCR specific for the methylated SERPIN-B5 promoter and the unmethylated UBE2B promoter. The precipitations were subsequent used to assess
the methylation at the human MCTS2,INPP5F_V2 and NAP1L5 DMRs. (B) Using the same PCR primer combinations, allelic-ChIP was performed
on human placental cell lines (for clarity, only ChIP-bound fractions are shown). (C) qPCR on ChIP-bound material from the TCL1 cell
immunoprecipitations. Levels of precipitation are compared to the SNURF DMR (red line, equal to one).
Nucleic Acids Research, 2011, Vol. 39, No. 11 4583

(the approximate size of the MCTS2 and NAP1L5 DMRs),
despite strong H3K4me2 enrichment. Together, these data
suggest that these repressive marks are not present, or very
low, in these two regions.
DISCUSSION
In this study, we have shown the paternal allele-specific
expression of INPP5F_V2,MCTS2 and NAP1L5 in a
wide range of human tissues. This is in contrast to the
mouse, where Inpp5f_v2 and Nap1l5 show spatial expres-
sion restricted to brain. We show that the TSSs for these
three genes are embedded in regions of differential DNA
methylation, similar to their mouse orthologues.
In the mouse, Mcts2 and Nap1l5, like Inpp5f_v2 are
organized such that the imprinted ‘intronic retrogene’
and its DMR/promoter reside in the intron of another
gene known as the ‘host’. In each case the retrogene
originated from an ancestoral gene on the X chromosome
some time in early eutheraian evolution, since Inppf5_v2,
Mcts2 and Nap1l5 are absent in marsupials (19).
Retrotransposition has also been linked to the imprinting
of the RB1 gene, however this event occurred much later
in mammalian evolution as only humans, chimpanzee and
rhesus monkeys, but not mice and rat have the processed
RB1/KIAA0649 pseudogene (25). A recent high-resolution
analysis of parent-of-origin allelic expression in mouse
brain has revealed that the host genes Herc3 and Inpp5f
show evidence for allele-specific alternative polyA choice
similar to that of H13 [A.J. Wood and R.J. Oakey,
unpublished data and (14,15)].
To assess whether the human orthologues MCTS2,
NAP1L5 and INPP5F_V2 are also associated with im-
printed host genes, we analyzed the allelic expression of
the host genes in a wide range of fetal tissues and term
placentas. We observe in all cases, that the host transcripts
are biallelically expressed. The genomic location of the
host gene exons are different. In the mouse, the polyA
signal for the paternally expressed H13d isoform maps
to within 100 bp of the Mcts2 DMR, whereas these two
features are separated by >7 kb in humans. Genomic re-
arrangements are more pronounced at the Nap1l5/
NAP1L5 locus, where Herc3b and Nap1l5 are separated
by 1.5 kb in the mouse, and by more that 39 kb in humans.
In addition, the mouse Herc3c isoform initiates from
within the Nap1l5 DMR, whereas the human promoter
for this isoform is adjacent to the HERC3B polyA
(Figure 1D). These differences in exon distribution could
explain the lack of host gene imprinting observed in
humans. We have previously proposed that transcription-
al interference may be involved in alternative polyA choice
at H13, due to the high expression of Mcts2 in brain
directly inhibiting the transcription of H13 on the
paternal allele in co-expressing cells. Alternative models,
including the recruitment of methylation-sensitive polyA
factors, or the association with the CTCF/cohesin
boundary complex to the unmethylated paternal allele of
the Mcts2, could result in similar allelic-termination of the
host genes on the maternal allele (14). For any of the
models, the imprinting of host gene transcripts may
require a close physical proximity of the host gene exons
with the retrogene, which is not observed in humans.
Genes flanking the retrogene-host pairs are biallelically
expressed
Many imprinted genes are clustered in the genome, such
that their expression is influenced by shared control
elements, such as DMRs. The imprinted retrogene-host
pairs are located outside of characterized clusters. To
confirm this, we assayed flanking genes for expression
status and found all to be biallelic, with the exception of
Abcg2 which is monoallelically expressed in the placentae
from reciprocal BxC and CxB sub-species intercrosses, but
not subject to imprinting.
It has recently become evident that non-imprinted
monoallelic expression can result from SNP-associated
DNA methylation (26). To assess whether the Abcg2
eQTL is due to monoallelic DNA methylation similar to
that described in humans, we showed that the promoter
CpG island of Abcg2 is fully unmethylated (Figure 2 and
data not shown) suggesting that another mechanism is
regulating the allelic expression. Recently, Brideau et al.
(27) reported that the AK006067 transcript next to the
imprinted Rasgrf1 gene is expressed >100-fold higher
from the C57BL/6 allele than the PWK allele. Both our
finding of the Abcg2 eQTL and the observations of
Brideau et al. (27), emphasize the necessity to study recip-
rocal F1 crosses.
Histone modification signatures at imprinted retrogene
DMRs
Recent studies have suggested that there is a link between
DNA and histone methylation at imprinted DMRs. A
comprehensive analysis of allelic histone modifications in
Dnmt3l
/+
conceptuses revealed that without oocyte-
derived DNA-methylation imprints, there is a dramatic
effect on the presence of repressive histone modifications,
with maternally DNA methylated DMRs adopting a
paternal epigenotype (7). We explored whether the chro-
matin at Nap1l5,Mcts2 and Inpp5f_v2 is associated with
the plethora of histone modifications known to be
enriched at DMRs. In agreement with our previous obser-
vations, we found that the DNA-methylated alleles of
each DMR are enriched for the repressive histone marks
H3K9me3, H4K20me3 and H2A/H4R3me2s. Unlike
these three modifications, H3K27me3 was not consistently
associated with the DNA-methylated allele, which agrees
with earlier chromatin studies on ICRs. At the Nap1l5
DMR, we confirm the previous observation of bivalent
chromatin in mouse embryos (7), with H3K4me2 and
H3K27me3 both enriched on the paternal allele. This
monoallelic bivalent domain behaves in a similar fashion
to the recently described monoallelic bivalent chromatin at
the Grb10 DMR (21). Like their non-imprinted bivalent
counterparts, these genes are associated with ‘poised’
lineage-specific transcription in mouse and human ES
cells (28). In brain, we observe absence of allelic enrich-
ment for H3K27me3, and this correlates with acquired
expression of Nap1l5, a mechanism comparable to
what we have reported previously at the Grb10 (21,22).
4584 Nucleic Acids Research, 2011, Vol. 39, No. 11

These observations suggest that monoallelic bivalent
chromatin could be a common mechanism conferring
brain-specific imprinted gene expression.
To confirm the conservation of these histone modifica-
tions at the regions we identified as DMRs in humans, we
performed ChIP on leukocytes and placenta cell lines. To
our surprise we did not find significant enrichment of the
repressive histone marks H3K9me3 and H4K20me3 on
the DNA-methylated allele. This uncoupled action of the
DNA-methylation machinery and the H3K9me3 and
H4K20me3 HMTs may be partially explained by the pro-
gressive decrease in CpG island size and CpG density at
the NAP1L5 and MCTS2 DMRs compared to other im-
printed DMRs in the genome (Supplementary Table S4).
Throughout mammalian evolution, the NAP1L5 and
MCTS2 DMRs have lost approximately half of their
CpG dinucleotides compared to mouse. Both DMRs
have diminished in size, with a reduction from >420 bp
to 216 bp for MCTS2. The human NAP1L5 DMR fails to
reach standard CpG island criteria (GC content >50%;
Obs CpG/Exp CpG >0.6; min length 200 bp). The loss in
CpG island size at the NAP1L5 DMR is due to a com-
bination of CpG deamination and the integration of
numerous CpG low-density repeat elements, including
DNA-MER115, LINE-1 and low-complexity CT-repeats
immediately downstream of the transcription start site.
The human and mouse genomes have recently been
shown to contain a similar number of CpG islands (29).
Our observation of an evolutionary loss of CpG density at
the NAP1L5 and MCTS2 DMRs goes against the general
trend. Additionally, for many imprinted DMRs analyzed
in humans (KvDMR1, GRB10,MEST,ZAC1,NDN,
GNAS EX1A,GNAS XL,PEG3,PEG10,NNAT and
IGF2R/AIR), the size of the CpG islands comprising the
DMRs are all larger in humans than in mouse
(Supplementary Table S4). The low-CpG density within
the promoters of NAP1L5 and MCTS2 may mean that
these discrete DMRs go unrecognized by the non-histone
proteins including the HMTs for H4K20me3 and
H3K9me3, respectively (8,12). Overall these observations
are in agreement with the recent studies suggesting that
DNA methylation at functional imprints require DNA
methylation before the acquisition of repressive histone
methylation (7), and that deficiencies in repressive
histone marks do not have a direct role in the regulation
of DNA methylation at ICRs (12,13,30).
CONCLUSIONS
In summary, we have shown that the human orthologues
of mouse imprinted retrogenes are paternally expressed in
a wide range of fetal tissues. In mice, the host genes are
subject to alternative polyadenylation, presumably as a
consequence of retrogene integrations that acquired im-
printing in proximity to weak polyA signals. In humans,
we show that retrogene promoters are subject to
allele-specific CpG methylation, but internal polyA sites
of host genes are situated further upstream of the DMRs
and thereby escape their influence. In mice, these DMRs
are associated with allelic repressive histone modifications.
At the mouse Nap1l5 promoter, monoallelic bivalent
chromatin i.e. the enrichment of both H3K4me2 and
H3K27me3 on the same allele, is associated with the
unmethylated paternal allele. In humans, an evolutionary
deterioration in CpG island size correlates with a lack
of allelic H3K9me3 and H4K20me3 precipitation at
NAP1L5 and MCTS2 DMRs indicating imprinted gene
expression in the absence of the an ICR-specific histone
signature.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank R. Schulz for critical reading of the article and
C. Landles for technical assistance with Southern blotting.
We thank K. Hata for Dnmt3l
/+
embryos on the BxJ
background and Dr M. Sullivan for the TCL cell lines.
FUNDING
Spanish Ministerio de Educacion y Ciencia (grant number
SAF2008-1578 to D.M.); Association Pour La Recherche
sur le Cancer (grant number 4980; 2010 to P.A.); La Ligue
contre le Cancer (grant ‘subvention comite’ Herault: 2010
to P.A.); CNRS ‘Projects for International Scientific
Cooperation’ (to P.A. and D.M.); Agence National de la
Recherche; ‘Institut National du Cancer’; Association for
International Cancer Research; ‘Ligue Contre le Cancer’
(to R.F.); Medical Research Council (to G.E.M.);
SPARKs; Wellbeing for Women (to G.E.M.); The
Wellcome Trust (to R.J.O., J.M.F. and M.C.); Ramon y
Cajal research fellowship (to D.M.); FPU studentship (to
A.G.A.). Funding for open access charge: Internal
funding.
Conflict of interest statement. None declared.
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