1 Laboratory of Molecular Immunoregulation, Basic Research Program,
SAIC-Frederick, National Cancer Institute, Frederick, MD 21702-1201, USA
2 Cancer and Developmental Biology Laboratory, National Cancer Institute,
Frederick, MD 21702-1201, USA
* Author for correspondence (e-mail: muegge{at}ncifcrf.gov)
Accepted 26 November 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Chromatin structure, DNA methylation, Gene imprinting, Lsh
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chromatin modifications that ensure the inheritance of the imprinting
pattern are first erased during gametocyte development and then
gender-specifically established either at late foetal stages or also after
birth in female germ cells (Constancia et
al., 1998; Verona et al.,
2003
; Delaval and Feil,
2004
). Epigenetic modifications are based on DNA methylation or
histone tail modifications that regulate the accessibility of chromatin. A
great deal of evidences points to CpG methylation as a major epigenetic
modification that commands the parental identity. Imprinted genes are
frequently associated with differentially methylated regions (DMRs) that are
established during gametocyte development and maintained with a high fidelity
throughout embryogenesis (Constancia et
al., 1998
; Mann et al.,
2000
). Mutational analysis of DMRs has supported their crucial
role in the mechanism of imprinting (Elson
and Bartolomei, 1997
; Wutz et
al., 1997
). CpG methylation, for example, can shape chromatin
structure by influencing histone modifications such as acetylation or
methylation levels (Bird,
2002
). This in turn can alter the accessibility of transcription
factors to their appropriate binding site and thus control gene expression.
CpG methylation may also directly interfere with the recruitment of DNA
binding factors to their target sites. For example, CpG methylation abolishes
binding of CTCF to the ICR (imprinting control center) of H19, a
differentially methylated region that controls H19 and Igf2
(insulin growth factor 2) gene expression
(Burgess-Beusse et al., 2002
).
The ICR is located about 2 to 4 kb upstream of the H19 gene
(Tremblay et al., 1997
).
Binding of CTCF in turn affects the organization of a `chromatin boundary'
that blocks the interaction of a downstream enhancer with the Igf2
promoter (Simpson et al.,
2002
). Ultimately, CTCF binding controls H19 gene
expression and suppresses Igf2 gene transcription. The importance of
CpG methylation for imprinting has also been demonstrated in
Dnmt1/ embryos that display reduced CpG
methylation and loss of imprinting at multiple genomic loci
(Li et al., 1993
;
Caspary et al., 1998
).
Lsh (lymphoid specific helicase), a member of the SNF2/helicase family of
chromatin remodeling proteins is an epigenetic regulator in mice
(Jarvis et al., 1996;
Geiman et al., 1998
;
Yan et al., 2003a
;
Huang et al., 2004
). Since Lsh
regulates DNA methylation (Dennis et al.,
2001
), we tested whether changes in CpG methylation levels in
Lsh/ mice can influence the mechanisms of
gene imprinting. As Lsh/ mice die at birth
(Geiman et al., 2001
), we
examined the maintenance of gene imprints in
Lsh/ embryos at day 17.5 of gestation. Among
six analyzed imprinted loci, mono-allelic expression was only disturbed in the
Cdkn1c (p57(Kip2)) gene by deletion of
Lsh. The bi-allelic expression of the Cdkn1c gene was
accompanied by a substantial reduction of CpG methylation at the 5' DMR
of the Cdkn1c gene. Furthermore, Lsh is specifically associated with
the promoter of Cdkn1c in wild-type cells. These results imply
independent control mechanisms for imprinted genes and suggest a crucial role
for Lsh and DNA methylation to execute the imprinting epigenetic program in a
locus-specific manner.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
RT-PCR analysis
Total RNA was isolated from day 17.5 embryo body tissues using RNAzol
(Tel-Test) and the expression patterns of individual genes were analyzed and
compared between Lsh+/+ and
Lsh/ embryos. Residual genomic DNA was
removed from total RNA with a DNase I (Invitrogen) treatment. Reverse
transcription was performed on 2 µg of total RNA using the Mu-MLV reverse
transcriptase kit (Ambion). Control reactions were prepared in parallel
without reverse transcriptase. The different transcripts were amplified from
1/40 of the reverse transcription reaction in the presence of each of the
specific primers. RFLP analysis was done as described above.
Histochemical staining for ß-galactosidase activity
We investigated ß-galactosidase gene expression by X-Gal
(5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) staining. Day 13.5
embryos were removed and rinsed with PBS. Embryos were fixed in fixative (0.2%
(v/v) glutaraldehyde, 1.5% (v/v) formaldehyde, 5 mM EGTA (pH 8.0), 2 mM
MgCl2, in PBS) for 30 minutes. The samples were washed three times
in buffer (2 mM MgCl2, 0.01% (w/v) sodium deoxycholate, 0.02% (v/v)
Nonidet P-40, in PBS). Staining was carried out at 30°C in the dark for 16
hours in the following buffer (1 mg/ml X-Gal, 5 mM
K4Fe(CN)6, 5 mM K3Fe(CN)6, in
PBS). For staining of tissue sections, embryos were immediately fixed in
liquid nitrogen; 5 µm thick coronal sections were mounted on a set of
gelatin-coated glass slides such that serial sections could be used for X-Gal
stains.
Bisulphite mutagenesis assay
An aliquot of 1 µg of genomic DNA was subjected to bisulphite treatment
using CpGenome DNA modification kit (Intergen Co) according to the
manufacturer's instructions. Primers were generated to match the DMRs of the
imprinting gene H19, Igf2r, Zac1 and Cdkn1c. The converted
DNA was subjected to methylation-specific nested PCR, using the following
primers (Table 1C) and
conditions: for H19 ICR (Tremblay
et al., 1997); for the Igf2r gene
(Lucifero et al., 2002
); for
Cdkn1c (Yatsuki et al.,
2002
). For outside primers the following PCR conditions were used:
cycling conditions were 3 minutes at 94°C, then 30 seconds at 94°C, 30
seconds at 58°C and 1 minute at 72°C for 35 cycles followed by a final
7-minute extension step at 72°C. For use of inside primers, 30 cycles were
used. The PCR products were separated in 1.5% agarose gels and purified using
the QIAEX II gel extraction kit (Qiagen). Amplified fragments were subcloned
into the pGEMT-Easy vector (Promega). Independent clones for each fragment
were sequenced by using the T7 primer.
Chromatin immunoprecipitation assay
In order to investigate the interaction between Lsh protein and chromatin,
we performed chromatin immunoprecipitation assays using a stable transfected
3T3 cell that expressed Flag-tagged Lsh under an inducible promoter
(Yan et al., 2003b).
Zeocin-resistant cells were induced with 100 pM mifepristone for induction of
the Lsh protein. Following cross-linking with culture medium containing 1%
formaldehyde at 37°C for 10 minutes and washing twice with ice-cold PBS
containing protease inhibitors (1 mM phenylmethyl sulfonyl fluoride (PMSF), 1
µg/ml aprotinin and 1 µg/ml peptatin A) cells were scraped off the
dishes and pelleted. The cells were resuspended in SDS lysis buffer (1% SDS,
10 mM EDTA, 50 mM Tris-HCl pH 8.1) with protease inhibitors for 10 minutes on
ice and sonicated four times for 30 seconds each at a power setting of 3.0
with the Sonicator 3000 (MISONIX) to get 100-1000 bp DNA fragments. The sample
was centrifuged to remove cell debris and diluted ten-fold in ChIP dilution
buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM, Tris-HCl pH 8.1,
167 mM NaCl, protease inhibitors). The supernatants were pre-cleared with 80
µl of a mixture of salmon sperm DNA-protein A agarose slurry (Upstate
Biotechnology). The slurry solution was centrifuged and the supernatants were
incubated with 2 µl of Flag M2 antibody (Sigma) or murine IgG1 as isotype
control with rotation overnight at 4°C. Then, 80 µl of salmon sperm
DNA-protein A agarose slurry was added and incubated for 1 hour. The beads
were washed several times, and the attached immune complexes were eluted with
a buffer containing 1% SDS and 0.1 M NaHCO3, followed by
reverse-crosslinking at 65°C for 4 hours. DNA was purified by proteinase K
digestion, phenol-chloroform extraction and ethanol precipitation. DNA was
resuspended in 100 µl of 1 xTE and 4 µl were used for PCR
analysis. The amplification profile was designed for 30 cycles using the same
primers described in the DMR polymorphism analysis
(Table 1D).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As shown in Fig. 1A, ß-galactosidase activity was detected in the tail of wild-type and Lsh heterozygous mice, when the lacZ allele was propagated from the paternal side. Similarly, the Lsh/ embryos displayed normal paternal expression pattern of the lacZ allele. When the lacZ allele was propagated from the maternal side, lacZ activity was completely suppressed in wild-type tails as well as in tissue derived from Lsh/ embryos. Thus silencing of the maternal allele was not relieved in the absence of Lsh. In order to determine if Lsh deletion possibly resulted in a `leaky' phenotype and could affect imprinting in selected tissues, whole embryo stains for ß-galactosidase activity were performed. As shown in Fig. 1B, several wild-type tissues expressed the paternal lacZ allele (such as the neural tube, somites, sympathetic ganglia, distal second brachial arch, and telencephalic vesicles, skeletal muscle, kidney, liver, lung, brain, and heart), and tissues from the Lsh/ embryo showed an indistinguishable staining pattern. In contrast, lacZ-tagged alleles that were maternally inherited exhibited complete suppression of the lacZ gene in all tissues examined. Similarly, Lsh/ embryos sustained an exclusively paternal expression pattern of the Zac1 locus, and did not show any signs of reactivation of the maternal allele in any tissue. Thus, despite global DNA hypomethylation in Lsh/ tissues, imprinting was not in general affected by the loss of Lsh.
|
|
|
First, we investigated part of the 3.7 kb-long DMR2 located within intron 2
of the Igf2r gene (Wutz et al.,
1997). Methylation of the DMR2 is maternally inherited and is
thought to control imprinting. In wild-type embryos, maternally derived clones
were almost completely methylated at all seven CpG sites within the examined
site, whereas the paternal allele was completely unmethylated
(Fig. 4). This DNA methylation
pattern was entirely preserved in the absence of Lsh. Thus neither did the
DMR2 of the Igf2r gene exhibit loss of DNA methylation, nor was any
change in the Igf2r gene expression pattern detectable, suggesting
that this imprinted locus was not affected by Lsh deletion.
|
The mouse Igf2 and H19 genes are located 70 kb apart on
chromosome 7, and demonstrate reciprocal imprinting status
(Fig. 2A). Only the paternal
allele of the Igf2 gene and only the maternal allele of the
H19 gene are expressed. A DMR upstream of the H19 gene is
essential for their parental allele-specific expression
(Tremblay et al., 1997). This
region contains conserved CTCF-binding sites involved in the establishment of
a `chromatin boundary' that regulates the imprinted expression of
Igf2 and H19. Recently, we studied DNA methylation of the
H19 locus at a 3.8 kb region comprising the ICR, examining about ten
methylation-sensitive HhaI sites by Southern analysis. Although
hypomethylation of some HhaI sites was observed, their precise location could
not be determined based on the high density of HhaI sites. Therefore a
conclusion as to whether CpG hypomethylation was indeed affecting CTCF binding
sites was not possible. Thus we examined in this study a 400 bp-long region of
the ICR that comprised two CTCF binding sites. (Note: Previous Southern
analysis had not shown any evidence of hypomethylation at the single HhaI site
located within this region.) Almost all 16 CpG sites were methylated on the
paternal allele, whereas the maternal allele was completely unmethylated
(Fig. 4). No significant
differences in the methylation pattern of the paternal allele comparing
wild-type and Lsh/ tissues were found. Thus
the similar methylation pattern in Lsh wild-type and Lsh-deleted samples
corresponded with the lack of expression changes in the Igf2 and
H19 genes.
Next, we examined the methylation changes at the Cdkn1c gene that
is maternally expressed (Hatada and Mukai,
1995) (Fig. 5). DNA
methylation analysis had identified several DMRs in the locus
(Yatsuki et al., 2002
). The
DMR (KvDMR1) of the Kcnq1ot1 (Lit1) promoter is located about 150 kb
downstream of the Cdkn1c gene. The KvDMR1 gets methylated in oocytes
and is unmethylated in sperm, and may represent an imprinting mark in this
domain. Loss of methylation and silencing of the Cdkn1c gene has been
implicated in patients with Beckman-Wiedeman syndrome
(Diaz-Meyer et al., 2003
). A
recent study reported that deletion of the KvDMR1 in mice results in
reactivation of the silenced paternal Cdkn1c allele
(Fitzpatrick et al., 2002
).
Two sites within the KvDMR1 were analyzed by bisulphite sequencing (known as
ICG8a and ICG8b) (Yatsuki et al.,
2002
), but no significant methylation differences were detectable
comparing Lsh+/+ and
Lsh/ samples. In contrast, the CpG
methylation pattern of the ICG5 site
(Yatsuki et al., 2002
) was
greatly altered in the absence of Lsh (Fig.
5). The ICG5 site is contained within a 5' DMR located in
the promoter upstream of the Cdkn1c gene and is largely methylated on
the paternal allele in wild-type controls. However,
Lsh/ samples had lost dramatically more than
half of the cytosine methylation. Thus the substantial decrease of CpG
methylation in the 5' DMR at the paternal allele is closely associated
with loss of paternal silencing and bi-allelic expression of the
Cdkn1c gene.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Various evidence points to DNA methylation as an important mechanism in
genomic imprinting. For example treatment of cell cultures with the
demethylating drug azacytidine abolishes the imprint of several genes
(El Kharroubi et al., 2001).
In addition, deletion of the major `maintenance' methyltransferase gene
Dnmt1 leads to global demethylation and a loss of imprinting at many
loci (Li et al., 1993
;
Caspary et al., 1998
). The
close association of CpG hypomethylation at the 5' DMR of the promoter
region, and the de-repression of the silent paternal Cdkn1c gene as
reported in this study give further support for a functional role of DNA
methylation in imprinting.
Two differentially methylated regions have been identified in the
Cdkn1c gene: KvDMR1 located in an intron within the Kcnq1
gene (about 150 kb downstream of the Cdkn1c gene) and 5' DMR
located in the Cdkn1c promoter region. The KvDMR1 is methylated on
the maternal allele, whereas the 5' DMR is methylated on the silenced
paternal allele. Furthermore the KvDMR1 site is critical for repression of the
silenced paternal allele, as deletion of the unmethylated paternal KvDMR1
results in bi-allelic expression of the embryo
(Fitzpatrick et al., 2002). In
contrast, deletion of the methylated KvDMR1 on the maternal allele had no
effect on Cdkn1c gene expression. However, CpG methylation must also
play a role in Cdkn1c silencing, since genomic hypomethylation caused
by deletion of the Dnmt1 gene resulted in bi-allelic expression of
Cdkn1c (Caspary et al.,
1998
). In this report we provide the first evidence that the
methylation of the 5' DMR is critical for paternal silencing, implying
that two mechanisms (suppression by the methylated 5' DMR as well as
umethylated KvDMR1) are critical for silencing of the paternal Cdkn1c
allele.
Although DNA methylation is an important mechanism in the control of
imprinting, not all loci are equally affected by methylation and other
chromatin-modifying mechanisms are involved, too. For example, in
Dnmt1/ mice, H19 and Cdkn1c are
activated by DNA hypomethylation, whereas Igf2, Igfr and
Kvlqt1 are silenced and the Mash2 gene appears unaffected
(Li et al., 1993;
Caspary et al., 1998
).
Likewise, uniparental murine embryonal fibroblasts that are treated with the
demethylating drug azacytidine show loss of imprinting and de-repression of a
few genes (H19, Cdkn1c, Peg3, Zac1), whereas other loci remain
unaffected (Grb10, Sgca, Snrpn, U2af1)
(El Kharroubi et al., 2001
).
Thus, distinct epigenetic mechanisms in addition to DNA methylation have been
postulated in the control of imprinting. Furthermore, in some cases the
establishment of imprinting may be independent of DNA methylation. For
example, in Dnmt3a/b-deleted ES cells, CpG methylation is lost at
several imprinted loci over time in culture
(Chen et al., 2003
).
Re-introduction of Dnmt3a/b transgenes leads to proper remethylation
of the paternal allele, but not the maternal allele. Therefore at least at
some loci, the imprinted memory is apparently stored as epigenetic
modification independent of CpG methylation. The 5' DMR (ICG5) of the
Cdkn1c is another example, since it is not methylated in germ cells,
but obtains differential methylation in somatic cells before day 7.5 of
gestation.
Since most DMRs such as the KvDMR1 and other DMRs analyzed in this study,
obtain their methylation pattern already in germ cells, they require only
maintenance of methylation during embryogenesis. Our results therefore suggest
that Lsh does not play a general role in maintenance of methylation, since
deletion of the major maintenance methyltransferase Dnmt1, in
contrast to Lsh deletion, leads to loss of methylation and imprinting
at many loci (Li et al., 1993;
Caspary et al., 1998
). Instead,
the specific effect of Lsh on the Cdkn1c gene is consistent with the
hypothesis that Lsh may play a role in de novo methylation because only the
5' DMR of the Cdkn1c gene acquires methylation in the embryo
and not in germ cells. Lsh may promote recruitment and association of de novo
DNA methyltransferases (such as Dnmt3a) to specific sites in the genome, or
alternatively may facilitate the DNA methyltransferase activity on nucleosomal
targets by remodeling chromatin and giving greater accessibility to hidden CpG
sites. The patchy loss of methylation at the 5' DMR of the
Cdkn1c gene (Fig. 5)
may reflect the inaccessibility of DNA methyltransferase in the absence of
Lsh, to the central portion of the nucleosomal DNA as opposed to the edges
that show greater probability of Dnmt exposure in vitro
(Okuwaki and Verreault, 2004
).
However, in order to test the hypothesis that Lsh plays an important role in
de novo methylation, different experimental systems are required. To examine a
general role in de novo methylation, the acquisition of methylation patterns
in integrated retroviral sequences in embryonal stem cells or in episomal
constructs should be tested (Okano et al.,
1999
; Hsieh,
1999
).
The human and mouse promoter regions of Cdkn1c share high
homology, however, only the murine CpG islands have been reported to show
differential methylation. Thus either the human CDKN1C gene may be
independent of methylation (and Lsh), or alternatively, Lsh may participate in
the imprinting control of the human gene, but independently of CpG
methylation. We have previously shown that Lsh also controls
post-translational modifications such as histone acetylation or methylation
levels (Yan et al., 2003; Huang et al.,
2004). However, further analysis of genomic imprinting control at
the human CDKN1c gene has to be performed in order to determine the
role of Lsh.
Loss of imprinting has been implicated in the origin of sporadic cancers
and human inherited syndromes that are cancer prone
(Reik and Walter, 2001;
Paulsen and Ferguson-Smith,
2001
; Feinberg et al.,
2002
). A subset of patients with Beckwith-Wiedemann syndrome that
are prone to childhood malignancies show a functional mutation in the
Cdkn1c gene. We report here the hypomethylation at the
Cdkn1c promoter correlated with bi-allelic expression. Since
Cdkn1c is a cell cycle inhibitor its role has been largely implicated
as a tumor suppressor gene whose loss of function promotes growth and tumor
progression. However, a number of tumors have been reported that do not show
silencing, but instead show overexpression of the Cdkn1c gene
(Hartmann et al., 2000
;
Lai et al., 2000
;
Ito et al., 2002
). For
example, a subset of patients with head and neck cancers, or patients with
hepatoblastoma exhibit an upregulation of Cdkn1c gene expression with
reactivation of the paternal allele, and frequent loss of heterozygosity of
the maternal gene. Furthermore, some patients with Wilms tumor show paternal
expression of Cdkn1c and loss of heterozygosity of the maternal
region. Though Cdkn1c is a cell cycle inhibitor, it interacts with
transcription factors (such as MyoD) and proteins of the
c-Jun/stress-activated kinase pathway
(Chang et al., 2003
;
Reynaud et al., 2000
). Thus
inhibition of the UV-or stress-induced apoptotic pathway Cdkn1c may
contribute to cancer progression or therapy resistance of some tumors.
Investigating Lsh's unique contribution to the epigenetic regulation at distinct imprinted loci should help our understanding of the multiple mechanisms that control imprinting, and their role in pathogenetic processes such as cancer.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartolomei, M. S. and Tilghman, S. M. (1997). Genomic imprinting in mammals. Annu. Rev. Genet. 31,493 -525.[CrossRef][Medline]
Bird, A. (2002). DNA methylation patterns and
epigenetic memory. Genes Dev.
16, 6-21.
Burgess-Beusse, B., Farrell, C., Gaszner, M., Litt, M., Mutskov,
V., Recillas-Targa, F., Simpson, M., West, A. and Felsenfeld, G.
(2002). The insulation of genes from external enhancers and
silencing chromatin. Proc. Natl. Acad. Sci. USA
99 Suppl. 4,16433
-16437.
Caspary, T., Cleary, M. A., Baker, C. C., Guan, X. J. and
Tilghman, S. M. (1998). Multiple mechanisms regulate
imprinting of the mouse distal chromosome 7 gene cluster. Mol.
Cell. Biol. 18,3466
-3474.
Chang, T. S., Kim, M. J., Ryoo, K., Park, J., Eom, S. J., Shim,
J., Nakayama, K. I., Nakayama, K., Tomita, M., Takahashi, et al.
(2003). p57KIP2 modulates stress-activated signaling by
inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase.
J. Biol. Chem. 278,48092
-48098.
Chen, T., Ueda, Y., Dodge, J. E., Wang, Z. and Li, E.
(2003). Establishment and maintenance of genomic methylation
patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol.
Cell Biol. 23,5594
-5605.
Constancia, M., Pickard, B., Kelsey, G. and Reik, W.
(1998). Imprinting mechanisms. Genome
Res. 8,881
-900.
Delaval, K. and Feil, R. (2004). Epigenetic regulation of mammalian genomic imprinting. Curr. Opin. Genet. Dev. 14,188 -195.[CrossRef][Medline]
Dennis, K., Fan, T., Geiman, T., Yan, Q. and Muegge, K.
(2001). Lsh, a member of the SNF2 family, is required for
genome-wide methylation. Genes Dev.
15,2940
-2944.
Diaz-Meyer, N., Day, C. D., Khatod, K., Maher, E. R., Cooper,
W., Reik, W., Junien, C., Graham, G., Algar, E., Der Kaloustian, V. M. and
Higgins, M. J. (2003). Silencing of CDKN1C (p57(KIP2)) is
associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome.
J. Med. Genet. 40,797
-801.
El Kharroubi, A., Piras, G. and Stewart, C. L.
(2001). DNA demethylation reactivates a subset of imprinted genes
in uniparental mouse embryonic fibroblasts. J. Biol.
Chem. 276,8674
-8680.
Elson, D. A. and Bartolomei, M. S. (1997). A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting. Mol. Cell Biol. 17,309 -317.[Abstract]
Fan, T., Yan, Q., Huang, J., Austin, S., Cho, E., Ferris, D. and
Muegge, K. (2003). Lsh-deficient murine embryonal fibroblasts
show reduced proliferation with signs of abnormal mitosis. Cancer
Res. 63,4677
-4683.
Feinberg, A. P., Cui, H. and Ohlsson, R. (2002). DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Semin. Cancer Biol. 12,389 -398.[CrossRef][Medline]
Fitzpatrick, G. V., Soloway, P. D. and Higgins, M. J. (2002). Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1. Nat. Genet. 32,426 -431.[CrossRef][Medline]
Geiman, T. M. and Muegge, K. (2000). Lsh, an
SNF2/helicase family member, is required for proliferation of mature T
lymphocytes. Proc. Natl. Acad. Sci. USA
97,4772
-4777.
Geiman, T. M., Durum, S. K. and Muegge, K. (1998). Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh). Genomics 54,477 -483.[CrossRef][Medline]
Geiman, T. M., Tessarollo, L., Anver, M. R., Kopp, J. B., Ward, J. M. and Muegge, K. (2001). Lsh, a SNF2 family member, is required for normal murine development. Biochim. Biophys. Acta 1526,211 -220.[Medline]
Hartmann, W., Waha, A., Koch, A., Goodyer, C. G., Albrecht, S.,
von Schweinitz,. D. and Pietsch, T. (2000). p57(KIP2) is not
mutated in hepatoblastoma but shows increased transcriptional activity in a
comparative analysis of the three imprinted genes p57(KIP2), IGF2, and H19.
Am. J. Pathol. 157,1393
-1403.
Hatada, I. and Mukai, T. (1995). Genomic imprinting of p57KIP2, a cyclin-dependent kinase inhibitor, in mouse. Nat. Genet. 11,204 -206.[Medline]
Hsieh, C. L. (1999). In vivo activity of murine
de novo methyltransferases, Dnmt3a and Dnmt3b. Mol. Cell
Biol. 19,8211
-8218.
Huang, J., Fan, T., Yan, Q., Zhu, H., Fox, S., Isaaq, H. J.,
Best, L., Gangi, L., Munroe, D. and Muegge, K. (2004). Lsh,
an epigenetic guardian of repetitive elements. Nucleic Acids
Res. 32,5019
-5028.
Ito, Y., Yoshida, H., Nakano, K., Kobayashi, K., Yokozawa, T., Hirai, K., Matsuzuka, F., Matsuura, N., Kuma, K. and Miyauchi, A. (2002). Expression of p57/Kip2 protein in normal and neoplastic thyroid tissues. Int. J. Mol. Med. 9, 373-376.[Medline]
Jarvis, C. D., Geiman, T., Vila-Storm, M. P., Osipovich, O., Akella, U., Candeias, S., Nathan, I., Dururm, S. K. and Muegge, K. (1996). A novel putative helicase produced in early murine lymphocytes. Gene 169,203 -207.[CrossRef][Medline]
Lai, S., Goepfert, H., Gillenwater, A. M., Luna, M. A. and El
Naggar, A. K. (2000). Loss of imprinting and genetic
alterations of the cyclin-dependent kinase inhibitor p57KIP2 gene in head and
neck squamous cell carcinoma. Clin. Cancer Res.
6,3172
-3176.
Li, E., Beard, C. and Jaenisch, R. (1993). Role for DNA methylation in genomic imprinting. Nature 366,362 -365.[CrossRef][Medline]
Lucifero, D., Mertineit, C., Clarke, H. J., Bestor, T. H. and Trasler, J. M. (2002). Methylation dynamics of imprinted genes in mouse germ cells Genomics 79,530 -538.[CrossRef][Medline]
Mann, J. R., Szabo, P. E., Reed, M. R. and Singer-Sam, J. (2000). Methylated DNA sequences in genomic imprinting. Crit. Rev. Eukaryot. Gene Expr. 10,241 -257.[Medline]
Okano, M., Bell, D. W., Haber, D. A. and Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99,247 -257.[Medline]
Okuwaki, M. and Verreault, A. (2004).
Maintenance DNA methylation of nucleosome core particles. J. Biol.
Chem. 279,2904
-2912.
Paulsen, M. and Ferguson-Smith, A. C. (2001). DNA methylation in genomic imprinting, development, and disease. J. Pathol. 195,97 -110.[CrossRef][Medline]
Piras, G., El Kharroubi, A., Kozlov, S., Escalante-Alcalde, D.,
Hernandez, L., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. and Stewart, C.
L. (2000). Zac1 (Lot1), a potential tumor suppressor gene,
and the gene for epsilon-sarcoglycan are maternally imprinted genes:
identification by a subtractive screen of novel uniparental fibroblast lines.
Mol. Cell Biol. 20,3308
-3315.
Reik, W. and Walter, J. (2001). Genomic imprinting: parental influence on the genome. Nat. Rev. Genet. 2,21 -32.[CrossRef][Medline]
Reynaud, E. G., Leibovitch, M. P., Tintignac, L. A., Pelpel, K.,
Guillier, M. and Leibovitch, S. A. (2000). Stabilization of
MyoD by direct binding to p57(Kip2). J. Biol. Chem.
275,18767
-18776.
Simpson, M., West, A. and Felsenfeld, G.
(2002). The insulation of genes from external enhancers and
silencing chromatin. Proc. Natl. Acad. Sci. USA
99 Suppl. 4,16433
-16437.
Smith, R. J., Arnaud, P., Konfortova, G., Dean, W. L., Beechey, C. V. and Kelsey, G. (2002). The mouse Zac1 locus: basis for imprinting and comparison with human ZAC. Gene 292,101 -112.[CrossRef][Medline]
Spengler, D., Villalba, M., Hoffmann, A., Pantaloni, C.,
Houssami, S., Bockaert, J. and Journot, L. (1997). Regulation
of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein
expressed in the pituitary gland and the brain. EMBO
J. 16,2814
-2825.
Taniguchi, T., Okamoto, K. and Reeve, A. E. (1997). Human p57(KIP2) defines a new imprinted domain on chromosome 11p but is not a tumour suppressor gene in Wilms tumour. Oncogene 14,1201 -1206.[CrossRef][Medline]
Tremblay, K. D., Duran, K. L. and Bartolomei, M. S. (1997). A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development. Mol. Cell Biol. 17,4322 -4329.[Abstract]
Verona, R. I., Mann, M. R. and Bartolomei, M. S. (2003). Genomic imprinting: intricacies of epigenetic regulation in clusters. Annu. Rev. Cell Dev. Biol. 19,237 -259.[CrossRef][Medline]
Wutz, A., Smrzka, O. W., Schweifer, N., Schellander, K., Wagner, E. F. and Barlow, D. P. (1997). Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 389,745 -749.[CrossRef][Medline]
Yan, Q., Huang, J., Fan, T., Zhu, H. and Muegge, K.
(2003a). Lsh, a modulator of CpG methylation, is crucial for
normal histone methylation. EMBO J.
22,5154
-5162.
Yan, Q., Cho, E., Lockett, S. and Muegge, K.
(2003b). Association of Lsh, a regulator of DNA methylation, with
pericentromeric heterochromatin is dependent on intact heterochromatin.
Mol. Cell Biol. 23,8416
-8428.
Yatsuki, H., Joh, K., Higashimoto, K., Soejima, H., Arai, Y.,
Wang, Y., Hatada, I., Obata, Y., Morisaki, H., Zhang, Z., Nakagawachi, T.,
Satoh, Y. and Mukai, T. (2002). Domain regulation of
imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5:
large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative
imprinting control region. Genome Res.
12,1860
-1870.
Related articles in Development: