a nonimprinted prader–willi syndrome (pws)-region gene regulates a different chromosomal domain in...
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Genomics 85 (20
A nonimprinted Prader–Willi Syndrome (PWS)-region gene regulates a
different chromosomal domain in trans but the imprinted PWS
loci do not alter genome-wide mRNA levels
Mihaela Stefana, Toni Portisb, Richard Longneckerb, Robert D. Nichollsa,*
aDepartment of Psychiatry, and Department of Genetics, Center for Neurobiology and Behavior, University of Pennsylvania, 415 Curie Boulevard,
Philadelphia, PA 19104-6140, USAbDepartment of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Ward 6-231, 303 East Chicago Avenue,
Chicago, IL 60611, USA
Received 22 November 2004; accepted 5 February 2005
Abstract
Prader–Willi syndrome (PWS) is a complex neurobehavioral disorder that results from loss of function of 10 clustered, paternally
expressed genes in a 1.5-Mb region of chromosome 15q11–q13. Many of the primary PWS region genes appear to have nuclear RNA
regulatory functions, suggesting that multiple genetic pathways could be secondarily affected in PWS. Using a transgenic mouse model of
PWS (TgPWS) with an ¨4-Mb chromosome 7C deletion of paternal origin that models the neonatal phenotype of the human syndrome we
compared by oligonucleotide microarrays expression levels of ¨12,000 genes and ESTs in TgPWS and wild-type brain. Hybridization data
were processed with two distinct statistical algorithms and revealed a dramatically reduced expression of 4 imprinted genes within the
deletion region in TgPWS mice, with 2 nonimprinted, codeleted genes reduced twofold. However, only 3 genes outside the deletion were
significantly altered in TgPWS mouse brain, with ¨1.5-fold up-regulation of mRNA levels. Remarkably, these genes map to a single
chromosome domain (18B3), and by quantitative RT-PCR we show that 8 genes in this domain are up-regulated in TgPWS brain. These
18B3 genes were up-regulated in an equivalent manner in Angelman syndrome mouse (TgAS) brain, which has the same deletion but of
maternal origin. Therefore, the trans-regulation of the chromosome 18B3 domain is due to decreased expression of a nonimprinted gene
within the TgPWS/AS mouse deletion in mouse chromosome 7C. Most surprisingly, since 48–60% of the genome was screened, it appears
that the imprinted mouse PWS loci do not widely regulate mRNA levels of other genes and may regulate RNA structure.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Disease mechanisms; Gene expression; Microarray; Quantitative mRNA transcription; Transregulation
Prader–Willi syndrome (PWS) is a complex devel-
opmental and multisystem disorder affecting ¨1/15,000
live births [1,2]. Clinically, PWS is characterized by a
two-stage natural history. PWS infants have failure to
thrive with muscle hypotonia, genital hypoplasia, respira-
tory problems, and feeding difficulties due to poor suck
and swallowing reflexes [1,3], but between 2 and 4 years
of age obesity sets in as a consequence of an uncon-
trolled eating behavior [4]. In addition, PWS subjects
0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygeno.2005.02.004
* Corresponding author. Fax: +1 215 898 0273.
E-mail address: robertn@mail.med.upenn.edu (R.D. Nicholls).
have an abnormal body composition with decreased
muscle mass and an increase in adiposity even prior to
onset of obesity, a lowered metabolic rate, short stature,
and mild to moderate mental retardation with behavioral
disorders [1–5]. To date, most of the hormonal and
metabolic abnormalities reported in PWS patients, inclu-
ding growth hormone (GH) and insulin-like growth factor
(IGF-1) deficiencies, have been assumed to be associated
with hypothalamic dysfunction but this is as yet
unproven.
PWS is caused by absence of expression of a cluster
of paternally expressed genes in chromosome 15q11–
q13, by paternal chromosomal deletion, maternal unipar-
05) 630 – 640
YGENO-07463; No. of pages: 11; 4C:
M. Stefan et al. / Genomics 85 (2005) 630–640 631
ental disomy (UPD), or imprinting defect mechanisms
[6]. De novo 4-Mb deletions account for ¨70% of PWS
cases [6–8], and span the 2-Mb imprinted domain and
several nonimprinted genes. There are 10 known pater-
nally expressed loci potentially involved in PWS etiology
(Fig. 1A). This includes NDN, encoding NECDIN, a
MAGE protein family member proposed to act as a
growth suppressor and antiapoptotic protein in postmitotic
neurons [11–13]. While NECDIN was initially described
as a nuclear protein [14,15] able to bind to specific DNA
sequences [16], recent data suggest a primarily cytoplas-
mic localization [13]. MAGEL2 is a related MAGE
family member of unknown function but with several
potential nuclear localization signals and is highly
expressed in the hypothalamus and other brain regions
[17,18]. A third locus, MKRN3, encodes a putative
ribonucleoprotein [19]. In addition, the complex IC–
SNURF –SNRPN –snoRNA polycistronic locus codes for
several functions including a cis-acting regulatory region
(imprinting center or IC), two nuclear proteins (SNURF
and SmN) [20,21], five classes of box C/D small
nucleolar RNA (snoRNA) [9,22,23], and UBE3A-AS
antisense transcript [23,24]. SmN is a spliceosomal
protein expressed primarily in brain, where it replaces
the otherwise constitutively expressed SmBV/B proteins.
In the absence of SmN in PWS patients and mice, SmBV/B levels increase significantly, which may largely
compensate for splicing functions [25]. SNURF is rich
in arginine residues and hence might bind RNA or have
an unknown function [21]. Each brain-specific snoRNA
in the PWS region lacks the usual rRNA complementar-
ity and may target cellular mRNAs for methylation or
alternative splicing [9,22,23]. Combined, these data
suggest that many of the ‘‘PWS primary genes’’ have
transcriptional or RNA regulatory functions and hence
Fig. 1. Genetic structure of (A) human chromosome 15q11–q13 and (B) the ho
flanked by PWS/AS common deletion breakpoints (BP, zigzag lines) is shown. The
[9]. The TgPWS/AS deletion (del) in mouse 7C [10,26] is equivalent to class I del
are circles, genes; black, paternal; gray, maternal; open, biparental; IC, imprinting c
of genes; *, mouse PWS/AS-region genes present on the MG-U74Av2 array; bar
that whole pathways of secondary and perhaps farther
downstream genes could be defective in PWS [5].
Human chromosome 15q11–q13 is homologous to the
central part of mouse chromosome 7, with conservation of
most gene content and imprinted features [6] (Fig. 1B).
Since mouse 7C imprinted loci lack a homolog to the
human HBII-438 snoRNA but have gained the retrotrans-
posed Frat3 gene [26], these are unlikely to play a
significant role in PWS. To date, targeted deletion studies
have not linked any individual mouse PWS-region gene to
any major clinical feature of PWS [27]. For example, Ndn-
deficient mice have a respiratory deficit with partial
neonatal lethality [28–30], mice harboring intragenic
deletions in Snurf –Snrpn were phenotypically normal
[31,32] although behavioral studies have not been reported,
while paternal deletion from Snurf to Ube3a results in
growth retardation and ¨80% lethality before weaning
[32]. In contrast, PWS mouse models with UPD, an
imprinting defect, or a paternally derived chromosomal
deletion share a very similar phenotype including hypo-
tonia, respiration difficulties, and failure to thrive, with
early postnatal lethality, modeling the first stage of the
human syndrome [6,10,31].
Given the neurobehavioral phenotype of PWS and the
putative regulatory features of many of the PWS-region
genes that are predominantly expressed in brain [33,34],
we have examined brain gene expression profiles in
transgenic deletion PWS (TgPWS) vs wild-type (WT)
mice at postnatal day 1 (P1) to identify candidates for
downstream gene pathways. Imprinted and nonimprinted
genes within the TgPWS mouse deletion were reduced in
TgPWS mouse brain, as expected, whereas there was
minimal change in global gene expression. Intriguingly,
expression of a cluster of eight genes outside the deletion
from chromosome 18B3 was significantly up-regulated in
mologous mouse chromosome 7C. The human 4-Mb 15q11–q13 region
HBII-85 and HBII-52 snoRNAs are repeated 24 and 47 times, respectively
etions in PWS and AS that occur in ¨40% of deletion patients [8]. Symbols
enter; cen, centromere; tel, telomere; line arrows, transcriptional orientation
at bottom, LMP2A transgene (Tg)-induced PWS/AS del mouse model [10].
M. Stefan et al. / Genomics 85 (2005) 630–640632
TgPWS brain as well as in mice with maternal inheritance
of the same deletion (an Angelman syndrome, or AS,
mouse model). These results suggest coordinate regulation
of expression of the 18B3 domain in trans by a non-
imprinted PWS/AS-region gene product.
Results
Gene expression in TgPWS vs WT brain at P1
We examined total RNA expression levels in the TgPWS
deletion vs WT mouse brain at P1 in two groups consisting
of five animals each, using high-density oligonucleotide
arrays with probes for ¨12,000 genes and ESTs. P1 was
chosen because at this age TgPWS mice do not display the
physiological changes of severe failure to thrive that
Fig. 2. Microarray analysis of brain gene expression in TgPWS vs WT mice. Gen
the MG-U74Av2 array with ¨12,000 genes/ESTs. (A) To evaluate expression prof
was performed in MAS 5.0 and generated a total of 16 transcripts as potentially der
log ratio (TSD). (B) A two-class unpaired analysis (SAM) yielded 20 transcripts
Both MAS 5.0 and SAM identified as the most significant (*) decreases in TgPWS
appears twice on the array, see Table 1). Other significant decreases are nonimprinte
were identified by both statistical methods as significantly changed in TgPWS, with
(Tcerg1), leucyl-tRNA synthetase (Lars), and protein phosphatase 2A, regulatory
commence at or after P2 (M.S., H. Ji, R.A. Simmons, D.E.
Cummings, K.H. Kaestner, R.S. Ahima, M.I. Friedman,
R.D.N., manuscript in preparation), thereby avoiding detec-
tion of changes secondary to failure to thrive. To detect
significant changes in gene expression between the two
groups, hybridization data were analyzed using two software
packages. The first comparison analysis was carried out
using Affymetrix MicroArray Suite 5.0 (MAS 5.0). After
applying the defining filtering criteria and establishing a
consistency of change in the same direction for at least three
of five replicate pair experiments, we identified 16 tran-
scripts as potentially deregulated in TgPWS brain with a fold
�1.4 (Fig. 2A). The second comparison was performed
using Significance Analysis of Microarrays (SAM) and
generated 20 potentially abnormally expressed genes in
TgPWS vs WT mice with a median false discovery rate
(FDR) of �45% (D = 0.3) (Fig. 2B). As expected, combined
e expression was compared for five TgPWS and five WT males at P1 using
iles from Gene Chip arrays for TgPWS and WT mice, a comparison analysis
egulated in TgPWS vs WT mice. Values are presented as the means of signal
with potentially abnormal expression in TgPWS vs WT brain (FDR � 45).
brain the PWS-region imprinted genes within the deletion (black bars; Ndn
d genes within the TgPWS deletion (open bars). Only three other transcripts
¨1.5-fold increases (striped light gray bars): transcription elongation factor
subunit B, h isoform (PP2ABb).
Fig. 3. Representation of mRNA expression levels of Cyfip1 in TgPWS and
TgAS vs WT mice at P1 and P4. Relative quantitation by QRT-PCR of
Cyfip1 mRNA showed a twofold decreased expression in TgPWS and
TgAS vs WT mice at all tested postnatal days. All values are the means
from five (P1) or four animals (P4) T SD. *p � 0.05; **p � 0.001; ***p �0.0001, significant differences between WT and TgPWS or TgAS at the
indicated time points (independent samples t test).
M. Stefan et al. / Genomics 85 (2005) 630–640 633
analysis of the two datasets identified the PWS-region
imprinted genes (Mkrn3, Magel2, Ndn, Snurf–Snrpn) as
the most significant decreases in gene expression in TgPWS
mice (Figs. 2A and 2B; Table 1). In addition, two non-
imprinted genes (Herc2, Cyfip1) within the deletion dem-
onstrate an ¨2-fold decrease in expression in TgPWS pups
(Figs. 2A and 2B; Table 1), which is in agreement with a
prediction of 50% reduction for these genes in TgPWS brain.
These six genes constitute built-in ‘‘controls’’ that give great
confidence in the microarray data, including the reproduci-
bility (all five TgPWS vs all five WT mice) and sensitivity
both for genes that have a low level of expression (e.g.,
Mkrn3) and for those with a small change in expression level
between WT and TgPWS mice (e.g., Herc2).
To confirm the reliability and sensitivity of detecting
subtle changes (less than or equal to twofold) in gene
expression in TgPWS mice, mRNA levels of Cyfip1, a
nonimprinted gene within the TgPWS/AS deletion, were
quantified by quantitative real-time PCR (QRT-PCR) in
brain tissue at P1 and P4. Consistent with the microarray
and the expectation for a deleted gene, the average mRNA
expression was decreased by twofold for Cyfip1 in TgPWS
vs WT mice and equivalent results were found for TgAS
vs WT mice (Fig. 3). Furthermore, since Cyfip1 transcript
levels were normalized to Gapdh levels, these data
demonstrate that the levels of Gapdh are not changed by
parameters of phenotype or age in this analysis, justifying
the use of this control gene in the following studies.
Despite the sensitivity of the microarray experiments,
only 3 genes not in the TgPWS deletion were identified
by both statistical methods with a consistent difference in
expression between the TgPWS and the WT groups:
Tcerg1, PP2ABb, and Lars (Figs. 2A and 2B; Table 1).
All 3 genes exhibit up-regulation in TgPWS vs WT brain
Table 1
Changes in brain gene expression in TgPWS vs WT mice at P1 by
microarray identified by comparison analysis in both MAS 5.0 and SAM
GenBank Gene MAS (5.0) SAM
D76440 Ndn –274.3 p < 0.0001 0.008 q = 20%
AJ00236a Ndn –137.1 p < 0.0001 0.005 q = 20%
X60388 Snurf–Snrpn –26.9 p < 0.0001 0.02 q = 20%
AJ243608 Magel2 –26 p < 0.0001 0.03 q = 20%
U19106 Mkrn3 (Zfp127) –10.4 p < 0.0001 0.06 q = 20%
AF072697 Cyfip1 (Shyc) –1.8 p < 0.0001 0.5 q = 36.3%
AF061529 Herc2 –1.7 p < 0.0001 0.6 q = 36.3%
B023485 Tcerg1 (CA150b) 1.5 p = 0.0002 1.5 q = 36.3%
AI844089 Lars (LeuRS) 1.4 p < 0.0001 1.5 q = 36.3%
AW048155 PP2ABb (PR55B/b) 1.4 p = 0.001 1.5 q = 36.3%
For pair-wise comparison analysis (MAS 5.0), all microarray values are
presented as the ratio of mean expression fold changes for TgPWS vs WT.
The p values were computed for expression levels (Signal) of individual
genes in TgPWS vs WT using a t test for independent samples. For two-
class unpaired analysis (SAM), all microarray results are presented as fold
change for TgPWS vs WT. The q values (%) represent the lowest FDR at
which the gene is called significant.a TFIIH EST but coligated with Ndn.
with the same fold change, ¨1.5 (Table 1). Moreover,
these genes show the maximum consistency of change (5/
5 experiments for PP2ABb and Lars and 4/5 experiments
for Tcerg1) by MAS 5.0. Microarray results were
confirmed by QRT-PCR at P1 for all 3 genes (Table 2).
Intriguingly, Tcerg1, Lars, and PP2ABb are clustered
within a 700-kb domain in mouse chromosome 18B3 and,
based on Fisher_s exact test, the probability of colocaliza-
tion of 3 of 12,000 genes spotted on MG-U74Av2 (with
23 genes within chromosome 18B) is 1.79 � 10�7. This
observation suggests that these 3 genes are regulated as a
domain.
Identification of eight genes in chromosome 18B3 that are
deregulated in TgPWS mice
Protein phosphatase 2A (PP2A) comprises a family of
serine/threonine phosphatases composed of catalytic (C),
structural (A), and regulatory subunits (B) [35]. There are
a large number of regulatory B subunits that independ-
ently associate with the constitutive core of the enzyme
consisting of subunits A and C. The B family of subunits
(PR55) is encoded by four genes: Ba, Bh, Bg, and By,with two major splice variants for PP2ABb generated by
use of alternate promoters, Bb.1 and Bb.2 [36]. Since our
studies above examined the 3V end of PP2ABb, we
performed QRT-PCR to distinguish the Bb.1 and Bb.2mRNA isoforms. This demonstrated that the Bb.1 isoform
was up-regulated 1.5-fold in PWS brain at P1 (Fig. 4A;
Table 2) and 1.5-fold at P4 (Table 2). In contrast, Bb.2expression remains unchanged between TgPWS and WT
mice (Fig. 4A; Table 2).
Five other genes were identified in the chromosome
18B3 cluster based on analysis of genomic sequence (Fig.
4B) and were not present on the MG-U74Av2 array:
Pou4f3, encoding a member of the Brn-3 family of POU
transcription factors [37]; Rbm27, encoding an RNA
binding polypeptide; Gpr151, encoding a member of the
Table 2
Chromosome 18B3 gene expression in TgPWS vs WT mouse brain at E18, P1, and P4
Genea QRT-PCR (E18)b QRT-PCR (P1)c QRT-PCR (P4)b
WT TgPWS p TgPWS/
WT ratio
WT TgPWS p TgPWS/
WT ratio
WT TgPWS p TgPWS/
WT ratio
PP2ABbd 1.08 (T0.09) 1.48 (T0.28) 0.03 1.4 1.08 (T0.09) 1.48 (T0.29) 0.04 1.4 1.17 (T0.15) 1.64 (T0.12) 0.003 1.4
PP2ABb.1 0.93 (T0.13) 1.14 (T0.10) 0.04 1.2 1.15 (T0.16) 1.69 (T0.18) 0.001 1.5 1.06 (T0.05) 1.55 (T0.09) <0.0001 1.5
PP2ABb.2 0.94 (T0.07) 1.06 (T0.13) 0.1 1.1 1.28 (T0.33) 1.03 (T0.17) 0.2 0.8 0.94 (T0.12) 0.82 (T0.11) 0.2 1
Tcerg1 1.23 (T0.20) 1.65 (T0.31) 0.07 1.3 1.11 (T0.07) 1.56 (T0.29) 0.01 1.4 0.90 (T0.08) 1.24 (T0.10) 0.003 1.4
Lars 1.16 (T0.15) 1.44 (T0.32) 0.2 1.2 1.27 (T0.46) 1.97 (T0.28) 0.02 1.6 0.93 (T0.14) 1.23 (T0.16) 0.03 1.3
Pou4f3 1.62 (T0.56) 2.90 (T1.03) 0.008 1.8e 1.73 (T0.95) 1.85 (T0.91) 0.8 1e 1.00 (T0.16) 1.42 (T0.30) 0.003 1.4e
Rbm27 1.17 (T0.13) 1.76 (T0.16) 0.001 1.5 1.04 (T0.11) 1.55 (T0.15) 0.0004 1.5 0.99 (T0.14) 2.17 (T0.55) 0.006 2.1
Gpr151 1.06 (T0.31) 1.08 (T0.32) 0.9 1 1.02 (T0.21) 1.11 (T0.14) 0.3 1e 0.93 (T0.13) 1.53 (T0.23) 0.004 1.6
C330008R14Rik 1.00 (T0.22) 1.50 (T0.43) 0.01 1.5e 1.87 (T0.82) 2.21 (T0.94) 0.4 1.2e 1 (T0.24) 1.7 (T0.23) 0.006 1.7
Sh3rf2 0.82 (T0.21) 1.16 (T0.08) 0.02 1.4 1.00 (T0.40) 1.41 (T0.24) 0.01 1.4e 1.09 (T0.20) 1.25 (T0.18) 0.06 1.1e
For each developmental stage (E18, embryonic day 18; P1, P4, postnatal days 1 and 4), QRT-PCR data are presented as the means of expression fold changes
(TSD) (first two columns), with two-tailed p values (third column), and the ratio of mean fold changes for TgPWS vs WT (fourth column).a GenBank accession numbers are in Table 1, also AK07905 (PP2ABb.1), AK013600 (PP2ABb.2), S69352 (Pou4f3), XM-128924 (Rbm27), NM_181543
(Gpr151), AK021193 (C330008R14Rik), and NM_172966 (Sh3rf2).b TgPWS, n = 4; WT, n = 4.c TgPWS, n = 5; WT, n = 5.d Probe and primers are in the 3Vend of the gene and do not differentiate between the b.1 and the b.2 isoform.e QRT-PCR results are pooled from two repeated reactions.
M. Stefan et al. / Genomics 85 (2005) 630–640634
G-protein-coupled receptor family expressed in the central
nervous system, predominantly in the trabecular complex
[38,39]; C330008R14Rik, encoding a putative protein
with a PRELI/MSF1 domain; and Sh3rf2, encoding a
polypeptide with SH3 domains and a RING Zn-finger
domain. QRT-PCR analysis revealed no change in
expression levels of Pou4f3, Gpr151 , and C33-
0008R14Rik in TgPWS mouse brain at P1, but an
¨1.5-fold significantly increased expression of Rbm27
and Sh3rf2 (Table 2). To investigate further the expres-
sion changes for chromosome 18B3 candidate genes
during the failure-to-thrive period and in embryonic life
of the TgPWS mouse, mRNA expression of these genes
was determined by QRT-PCR at P4 and E18, respectively.
For the most part, the few genes showing discordant data
at any one age (e.g., Pou4f3 at P1, Sh3rf2 at P4) show
wide standard deviations in expression levels within
groups, possibly due to low levels of expression
(Supplemental Data Fig. S1). At E18, five genes exhibit
significant expression variance between TgPWS and WT
mice (PP2ABb.1, Pou4f3, Rbm27, Sh3rf2, C330008R14-
Rik), at P1 the number of deregulated genes is also 5
(PP2ABb.1, Tcerg1, Rbm27, Lars, Sh3rf2), and by P4 all
genes within this domain except for Sh3rf2 (and the
PP2ABb.2 isoform) were up-regulated in TgPWS vs WT
mice (Table 2; Fig. 4B).
A nonimprinted PWS/AS region gene is responsible for
deregulation of the 18B3 domain
The data to date suggested that a gene associated with
the LMP2A transgene insertion and deletion in TgPWS
mice [10] regulates in trans a chromosome 18B3 domain
of eight genes, but does not distinguish between an
imprinted (paternally expressed) and a nonimprinted
PWS-region gene as being causative nor whether this
could be an effect of the LMP2A transgene insertion. To
distinguish between the first two possibilities, mRNA
levels for genes within the 18B3 region were assessed by
QRT-PCR in brain tissue of TgAS vs WT mice at P1 and
P4 (Table 3). At P1, with the exception of Lars, the same
genes displaying up-regulation in TgPWS brain show
between 1.4- and 1.8-fold significantly increased mRNA
levels in TgAS vs WT brain. At P4, the number of
deregulated genes within the 18B3 region increases to 5
following a similar pattern as in TgPWS brain (Table 3,
Fig. 4B). These data rule out an imprinted gene in the
chromosome 18B3 effect in these mice.
To distinguish a role for a nonimprinted, deleted gene or
the LMP2A transgene, expression of three genes within the
18B3 domain that showed increased mRNA levels in
TgPWS and TgAS brain was assessed by QRT-PCR in
liver tissue from EALMP2A vs WT mice at P1. Liver was
chosen as control since EALMP2A mice do not have
detectable expression of the transgene in brain, but do have
significant LMP2A expression in B cells of neonatal liver.
All three genes (Tcerg1, Rbm27, Sh3rf2; Supplemental
Data Table S1) are significantly up-regulated in TgPWS
liver and one (Rbm27) in TgAS (the other two genes have a
large SD in expression levels, possibly due to low level of
expression; Supplemental Data Fig. S1). In contrast, no
significant expression variance for Tcerg1, Rbm27, and
Sh3rf2 was observed for EALMP2A and WT liver (Supple-
mental Data Table S1). Similarly, expression of PP2ABb,Tcerg1, Lars, Rbm27, and Sh3rf2 in P1 brain does not differ
between EALMP2A and WT mice (data not shown).
Therefore, the transgene does not affect chromosome
18B3 expression.
Fig. 4. Abnormal expression of chromosome 18 domain in TgPWS and TgAS mice. (A) Representation of mRNA expression levels of PP2ABb.1 and
PP2ABb.2 in TgPWS vs WT mice. Relative quantitation of expression of Bb.1 and Bb.2 isoforms was performed in two groups of five TgPWS males (open
bars) and five WT males (black bars) at P1 and in four TgPWS and four WT mice at E18 and P4 by QRT-PCR. The average mRNA expression for Bb.1 was
increased by 1.2-fold at E18 (*p � 0.001), 1.5-fold at P1 (**p � 0.001), and 1.5-fold at P4 (***p � 0.0001) in TgPWS vs WT, whereas no change in the
average expression of Bb.2 was detected. (B) Model for trans-regulation of a chromosome 18B3 domain by a nonimprinted mouse TgPWS/AS locus. A
nonimprinted gene present in the TgPWS/AS deletion region is hypothesized to encode a factor that activates specific regulatory DNA elements that inhibit
transcription at various developmental time points for C330008R14Rik, Sh3rf2, Lars, Rbm27, Pou4f3, Tcerg1, Gpr151, and PP2ABb.1, but not PP2ABb.2.Alternatively, the PWS/AS nonimprinted gene may control a gene pathway leading to a factor that regulates the 18B3 domain. In TgPWS and TgAS mice,
haploinsufficiency of the nonimprinted gene results in increased expression of the chromosome 18B3 target genes. For the eight genes in this domain, the first
and last exons (black boxes) and transcriptional orientation (horizontal arrows) are shown. Symbols used: +, activation; j or – , increase or no change in
expression level, respectively (black j or – , TgPWS; gray j or – , TgAS).
M. Stefan et al. / Genomics 85 (2005) 630–640 635
Candidate gene assessment in TgPWS brain
Genes that could be considered a priori as candidates
for expression changes in TgPWS mice were individually
inspected in the microarray data set, if present on the chip,
and either exhibited no change in TgPWS vs WT or were
not expressed at detectable levels (Supplemental Data
Table S2). This analysis identified a few notable examples,
such as Ube3a, which maps within the TgPWS/AS deletion
but with maternal-only expression in brain and, as expected,
does not change in TgPWS vs WT (mRNA signal levels:
WT 96, PWS 84). Another candidate, Snrpb, was identified
as unchanged by microarray (WT 1718, PWS 1780),
suggesting that the up-regulation in protein levels observed
in PWS patient and TgPWS mouse model brain [25] occurs
posttranscriptionally. Other candidates, including Gh and
Igf-1, had unchanged mRNA levels in TgPWS vs WT brain,
as did genes implicated in food intake and energy balance
regulation, such as agouti-related protein (Agrp) and
neuropeptide Y (Npy). Secretogranin II (SCG2), a gene
that exhibits strong down-regulation in clonal fibroblast cell
lines from a PWS patient who is mosaic for normal cells and
cells with maternal uniparental disomy 15 [40], showed no
difference in brain mRNA levels in the TgPWS mouse
model. Therefore, SCG2 is unlikely to be relevant to the
PWS phenotype.
Discussion
Minimal gene expression changes in brain of the TgPWS
mouse model
In this study we used oligonucleotide microarrays and
QRT-PCR to ascertain the primary transcriptional changes
in the brain of a PWS mouse model, prior to onset of the
severe neonatal failure-to-thrive phenotype. Our microarray
screen reliably identified 4 imprinted and 2 nonimprinted
Table 3
Chromosome 18B3 gene expression in TgAS vs WT mouse brain at P1 and P4
Genea QRT-PCR (P1)b QRT-PCR (P4)c
WT TgAS p TgAS/WT ratio WT TgAS p TgAS/WT ratio
PP2ABbd 1.04 (T0.03) 1.84 (T0.28) 0.001 1.8 1.13 (T0.12) 1.85 (T0.17) <0.0001 1.6
PP2ABb.1 0.95 (T0.05) 1.39 (T0.17) 0.003 1.4 1 (T0.17) 1.5 (T0.16) 0.001 1.5
PP2ABb.2 1.08 (T0.10) 1 (T0.12) 0.3 1 0.91 (T0.10) 0.88 (T0.11) 0.6 0.9
Tcerg1 1.12 (T0.19) 1.70 (T0.22) <0.0001 1.5e 1.02 (T0.14) 1.24 (T0.17) 0.008 1.2e
Lars 1.13 (T0.34) 1.39 (T0.46) 0.2 1.2e 1.04 (T0.57) 1.36 (T0.51) 0.4 1.3
Pou4f3 0.79 (T0.21) 1.03 (T0.27) 0.2 1.3 1.11 (T0.32) 1.50 (T0.46) 0.1 1.4
Rbm27 1.03 (T0.12) 1.51 (T0.10) 0.001 1.5 1.06 (T0.13) 1.41 (T0.19) 0.01 1.4
Gpr151 1.10 (T0.15) 1.14 (T0.22) 0.8 1 1.28 (T0.32) 2.16 (T0.25) 0.001 1.6
C330008R14Rik 1 (T0.2) 1.3 (T0.18) 0.1 1.3 0.86 (T0.15) 1.09 (T0.21) 0.09 1.2
Sh3rf2 1.3 (T0.27) 2.1 (T0.57) 0.03 1.6 0.8 (T0.14) 1.1 (T0.23) 0.03 1.3
For each developmental stage (P1, P4, postnatal day 1 and 4), QRT-PCR data are presented as the means of expression fold changes (TSD) (first two columns),
with two-tailed p values (third column), and the ratio of mean fold changes for TgAS vs WT (fourth column).a GenBank accession numbers are in Table 2.b TgAS, n = 4; WT, n = 4.c TgAS, n = 5; WT, n = 5.d Probe and primers are in the 3Vend of the gene and do not differentiate between the b.1 and the b.2 isoform.e QRT-PCR results are pooled from two or three repeated reactions.
M. Stefan et al. / Genomics 85 (2005) 630–640636
genes from within the ¨4-Mb deletion as reduced in
TgPWS mouse brain, illustrating the sensitivity and
reproducibility of this approach. Combined with other work
defining the deletion endpoints ([10,26]; M.S., K. Claiborn,
J.M. Greally, R.D.N, unpublished data), the TgPWS/AS
mouse model has a deletion of 13 known imprinted and 10
nonimprinted genes (Fig. 1B) that is equivalent to class I
deletions in PWS and AS [6–8]. An additional set of
clustered genes from a different chromosome was identified
by microarray analysis as deregulated in TgPWS mouse
brain with ¨1.5-fold increased expression and is discussed
below. Although additional candidate genes may be
identified when whole genome mRNA arrays become
available, we screened between 48 and 60% of the
expressed mouse genome [41]. Therefore, despite the
neonatal lethality of the TgPWS mouse model, we conclude
that very few genes have significantly altered brain
expression levels as a primary genetic change in TgPWS
mice.
That few genes have significant changes in expression
levels in the newborn TgPWS mouse brain was unexpected
and may have several alternative explanations. Due to the
small size of the hypothalamus, key genes might not be
detectable by microarray analysis of whole brain, even if
expression is altered. Some but not all hypothalamic-
specific genes were detectably expressed (Supplemental
Data Table S2). Nevertheless, the role of hypothalamus in
PWS is unknown and remains hypothetical, particularly as
the mouse PWS-region genes are expressed more widely in
the brain [6,33,34]. In addition, developmentally determined
gene expression changes may be more readily detected in
older mice. However, since physiological and metabolic
changes associated with failure to thrive become evident in
the TgPWS mouse beginning at P2, many mRNA changes
in older TgPWS pups will be secondary to glucose
homeostasis and/or hepatic energy metabolism impairments.
We previously observed alterations in the orexigenic Agrp
and anorexigenic Pomc mRNA levels in the hypothalamic
arcuate nucleus in P3 TgPWS mice using in situ hybrid-
ization [42], but in the current study Pomc levels were
below detection while Agrp had no change in P1 mice,
suggesting the change at P3 of the latter may be secondary
to failure to thrive in PWS. Another possible basis for
finding few genes with altered expression in TgPWS mice is
that the brain may be a secondary mediator of primary
changes in peripheral organs, despite the predominant
neuronal expression of PWS-region imprinted genes
[6,33,34].
Alternatively, the PWS-region genes may encode factors
that regulate mRNA modification or structure rather than
transcriptional levels. Indeed, SmN (encoded by Snrpn)
replaces SmBV/B in the core spliceosome in neurons and
may play a role in neuronal-specific mRNA alternative
splicing patterns [6,20,21]. Moreover, the C/D box snoR-
NAs in the PWS region may have roles in cellular RNA
modification (e.g., site-specific 2V-O-ribose methylation) or
alternative splicing [9,22,23]. Changes in modification and/
or splicing of target mRNAs may change transcript levels
only minimally or not at all. Nevertheless, alterations in
mRNA modification or splicing could subsequently lead to
significant changes in protein structure and/or expression.
Therefore, proteomics approaches in TgPWS mice will
complement future studies of splicing patterns both globally
and for candidate genes to provide a more comprehensive
picture of the molecular changes underlying the TgPWS
mouse neonatal phenotype.
Minimal gene expression changes in congenital and
acquired diseases
Similar to our study on a PWS mouse model,
surprisingly subtle transcriptional changes have also been
M. Stefan et al. / Genomics 85 (2005) 630–640 637
identified by microarray in mouse models for other
developmental and neurological syndromes. For example,
Mecp2 mutant mice, which exhibit phenotypic similarities
to Rett syndrome, display very few genes with signifi-
cantly changed brain expression despite the expected role
of MECP2 in modulating methylcytosine silencing of gene
expression [43]. Although genes with 1.5-fold increased
expression have been reliably identified from the trisomic
region of a Down syndrome (DS) mouse model, support-
ing a role for gene dosage in DS, no changes in gene
expression levels from other regions of this or other
chromosomes were identified [44]. Similarly, significant
expression changes for oxidative phosphorylation genes in
diabetic and prediabetic muscle are altered by only 0.2- to
0.5-fold [45–47]. Along with our data on TgPWS mice,
these findings imply significant relevance of minimal gene
expression changes for the molecular pathogenesis of
several congenital syndromes and acquired diseases.
Therefore, we suggest that major changes in expression
of genetic pathways in vivo may be limited to severe
pathological states such as cancer or embryonic lethal
conditions.
A PWS/AS-region nonimprinted gene trans-regulates a
cluster of chromosome 18B3 genes
Another intriguing finding from our experiments was
identification of a cluster of genes from chromosome
18B3 with coordinate abnormal expression in the TgPWS/
AS mouse model. The next genes centromeric (Sb140, 2
Mb) and telomeric (DpysI3, Eif3alpha, ¨0.8 Mb) to this
domain are unchanged in TgPWS by microarray (data not
shown). Indeed, a boundary to the coregulated 18B3 gene
cluster likely exists within the PP2ABb locus, since there
is up-regulation of the promoter (Bb.1) closest to the
cluster of genes that are also up-regulated, whereas the
considerably more distal promoter (Bb.2) is not affected in
either TgPWS or TgAS mice (see Fig. 4). Our data also
suggest a temporal regulation for genes in the 18B3
domain, since fewer genes have abnormal regulation in
TgPWS brain at E18 and at P1 than at P4 (Fig. 4B). A
largely similar expression pattern of the chromosome
18B3 genes was identified in the TgAS mouse model at
P1 and P4, excluding a role of a PWS imprinted locus in
the regulation of this domain. We also ruled out an effect
of the LMP2A transgene with an estimated ¨80 copies in
TgPWS [10] on chromosome 18B3 gene expression by
analysis of control EALMP2A transgenic mice that have
B-cell-lineage expression of LMP2A. Therefore, we
conclude that reduced expression of a PWS/AS-region
nonimprinted gene is responsible for the coordinated
deregulation of an eight-gene expression domain in
chromosome 18B3.
The finding of up-regulation of a cluster of genes from
chromosome 18B3 in both TgPWS and TgAS mouse brain
raises the question of whether deregulation of the
homologous genes from human chromosome 5q31 may
play any role in PWS and AS in the human. Identifying the
specific nonimprinted PWS/AS-region gene involved in the
trans-effect (see below) will determine whether this is
likely to be a conserved mechanism, and future studies of
gene expression on autopsy material from PWS and AS
patients can address whether equivalent molecular changes
do occur in human. Since PWS and AS deletion patients
share few overlapping phenotypic characteristics, other
than hypopigmentation associated with hemizygous dele-
tion of at least the OCA2 gene [6], it appears likely that
up-regulation of the 5q31 genes does not play a major
phenotypic role in PWS and AS. Nevertheless, the greater
neurobehavioral severity of AS compared to PWS may
mask otherwise shared clinical characteristics. Therefore,
clinical and molecular studies of individuals with 5q31
duplications and increased dosage of the same genes as
potentially up-regulated in PWS and AS may prove
revealing.
Based on the observed gene expression patterns, we
suggest a model by which coordinate regulation of the 18B3
domain involves a chromatin protein or RNA encoded by a
PWS/AS-region nonimprinted gene, which binds directly a
cis-silencing element in 18B3 and has domain-wide silenc-
ing effects (Fig. 4B). This genetic control mechanism would
be disrupted in TgPWS and TgAS deletion mice, with up-
regulation of the target genes in chromosome 18B3.
Alternatively, the PWS/AS-region nonimprinted gene may
encode a molecule that impacts a silencing mechanism
indirectly. As noted above, there are currently 10 known
nonimprinted genes within the TgPWS/AS deletion that are
candidates for the putative silencing factor. An additional
possibility is reduced level of nonimprinted Ube3a or
Atp10c expression in TgPWS and TgAS mouse brain,
although this was not detected by microarray for Ube3a.
Several of the nonimprinted loci encode receptors, trans-
porters, or channels (Gabrb3, Gabra5, Gabrg3, p/Oca2,
Nipa1, Nipa2, Atp10c, Chrna7) ([6,48,49]; M.S., K. Clai-
born, J.M. Greally, R.D.N, unpublished data) and appear
unlikely to be involved. Gcp5 encodes a component of the
g-tubulin complex required for microtubule nucleation at
the centrosome [50], while Herc2 putatively is involved in
protein trafficking and degradation [51,52], roles that can
likely be ruled out as direct, but indirect roles remain
possible. Cyfip1 encodes a protein that interacts with the
small GTPase Rac1, implicated in neuronal development
and maintenance [53], and also interacts with the fragile X
mental retardation 1 (FMR1) protein [54]. FMR1 is
implicated in negative regulation of translation, possibly
by functioning in RNAi- or miRNA-related pathways [55].
Ube3a encodes an E3 ubiquitin ligase involved in protein
degradation [6,56] and which can impact gene expression
through p53-, steroid hormone receptor-, and NFX-related
mechanisms [6,56]. It is also possible that additional
nonimprinted candidate genes in the PWS/AS domain await
discovery. Further studies making use of mouse models with
M. Stefan et al. / Genomics 85 (2005) 630–640638
specific gene mutations for candidate genes ([51,52]; L. Li,
S. Mertzer, R.D.N, D. Carpenter, E.M. Rinchik, D.K.
Johnson, Y. You, unpublished data) and/or mice that carry
radiation-induced chromosomal deletions spanning the p
locus [57,58] will likely identify the nonimprinted gene
responsible for the chromosome 18B3 trans effect. Addi-
tional studies of temporal changes in chromatin structure in
the 18B3 domain in TgPWS or TgAS mice will shed light
on the molecular mechanisms of coordinate trans-regulation
by a gene in the PWS/AS domain.
Materials and methods
Animals
The PWS/AS deletion mouse model has an LMP2A-
transgene-induced ¨4-Mb deletion (Fig. 1B) [10]. Maternal
transmission of the transgene leads to the TgAS mouse
model, with a viable, fertile phenotype and presumably with
similar mild neurobehavioral features compared to AS
knockout mouse models [6], as well as a late-onset obesity
(unpublished data). In contrast, transgene transmission
through males results in the TgPWS mouse phenotype.
Construction and characterization of additional LMP2A
transgenic mice with B-cell-lineage expression of LMP2A
(EALMP2A) have been described previously [59]. Brain
tissues from five TgPWS and five WT male CD-1 sibs at P1
were used for microarray studies. Brain and liver tissues
from at least four TgPWS, TgAS, and EALMP2A and
littermate WT mice were used for QRT-PCR experiments.
Brain gene expression was determined by QRT-PCR in
TgPWS vs WT mice at E18, P1, and P4, whereas for TgAS
vs WT and EALMP2A vs WT mice QRT-PCR was
performed at P1 and P4. Liver mRNA levels were evaluated
by QRT-PCR at P1 and P4 in all three mouse models. All
animals were bred and genotyped as described [10,59]. The
University of Pennsylvania and Northwestern Institutional
Animal Care and Use Committees approved all animal
experiments.
Microarray analysis
Total RNA was extracted using TRIzol (Invitrogen,
Carlsbad, CA, USA), treated with RQ1 RNase-free DNase
(Promega, Madison, WI, USA), and checked for integrity
by gel electrophoresis. Microarray hybridization and
staining were performed at the Penn Microarray Facility
using the Affymetrix Murine Genome U74Av2 array. This
array represents all sequences (¨6000) in the Mouse
UniGene database (build 74) that were functionally
characterized, with an additional ¨6000 EST clusters
also represented. Target preparation, hybridization, and
initial data analysis were carried out as described (http://
www.affymetrix.com/index.affx: GeneChip Analysis Tech-
nical Manual). To evaluate expression profiles from Gene
Chip arrays for TgPWS and WT mice, a comparison
analysis was performed in MAS 5.0 (Affymetrix, Santa
Clara, CA, USA) and Excel (Microsoft, Redmond, WA,
USA). Five biological replicates of the pair-wise compar-
ison were done using 10 microarrays, 1 for each of the P1
animals described above. The results were organized by
filtering out genes called No Change or Absent on both
Gene Chips, sorted by Change p value and Signal Log
Ratio, and then converted to Fold Change. An average fold
change derived from all pair-wise comparisons of �1.4 was
used as the cut-off for potential significant differences in
gene expression and only genes that showed a consistent
change in the same direction for at least three of five
replicate pair experiments were further considered.
Normalized microarray hybridization data were further
imported into and analyzed by SAM (http://www-stat-class.
stanford.edu/SAM/servlet/SAMServlet) to identify genes
with highly reproducible expression changes. SAM assigns
a score to each gene in a gene expression profile based on
its change in expression relative to the standard deviation
of repeated measurements for that gene [60]. A gene is
considered significantly changed if it exceeds an arbitrary
adjusted threshold (D). SAM calculates a FDR, which
represents the median percentage of genes that are
incorrectly identified as significant [60]. Two-class
unpaired analysis was applied to compare gene expression
profiles of five TgPWS and five WT samples. For D = 0.3,
the permutated dataset generated 20 significant genes with
FDR � 45%.
QRT-PCR
Total RNA was isolated as above and cDNA was
synthesized using random hexamers with the Superscript
First-Strand Synthesis System (Invitrogen). For TgPWS vs
WT (P1), RT-PCR mRNA quantification was carried out
on the same samples as for microarray. QRT-PCR was
performed on an Applied Biosystems, Inc. (ABI) (Foster
City, CA, USA) Prism 7000 sequence detection system
(version 1.6) using SYBR Green dye. Matching primers
for each gene (Supplemental Data Table S3) were
designed using the Primer Express program (ABI). Relative
quantification of gene expression data was performed using
a comparative CT method (http://home.appliedbiosystems.
com/: User Bulletin 777802-002). Gapdh was used as an
endogenous control for comparison of test gene expression,
with DCT values calculated for each sample. One WT DCT
value for each developmental time point (E18, P1, P4) was
set as a calibrator value of 1 and all WT and TgPWS or
TgAS values from the same time point were normalized to
this. The DDCT values were then calculated by subtracting
the calibrator value from each DCT value. These were then
converted to expression fold changes (TSD) as described
(ABI). Mean expression fold changes (TSD) for WT and
TgPWS or TgAS samples and two-tailed p statistics were
calculated using a t test for independent samples (Analyze-
M. Stefan et al. / Genomics 85 (2005) 630–640 639
It, Microsoft Excel). A ratio of mean fold changes was then
determined for the two groups (TgPWS or TgAS vs WT).
BAC contig assembly
The chromosome 18 contig data were generated by stand-
ard Ensembl (http://www.ensembl.org/Mus_musculus/) and
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) analyses.
Acknowledgments
We thank Brande Latney for technical assistance, Dr.
Don Baldwin for assistance and training with the microarray
experiments, Dr. Sue Keilbaugh for training in QRT-PCR
methodology, Melissa Fazzari, Drs. Hong Ji and John
Tobias for helpful discussions, and Drs. John M. Greally
and Don Baldwin for comments on the manuscript. This
work was supported by the National Institutes of Health
(HD31491 and HD36079 to R.D.N.). R.L. is supported by
Public Health Service Grants CA62234, CA73507, and
CA93444 from the National Cancer Institute and DE13127
from the National Institute of Dental and Craniofacial
Research. M.S. is supported by an award from the American
Heart Association. T.P. is a Special Fellow of the Leukemia
and Lymphoma Society of America.
Appendix A. Supplementary data
Supplementary data for this article may be found on
ScienceDirect.
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