Transcript 926 presentation

CTCF maintains differential
methylation at the Igf2/H19 locus
• Christopher J. Schoenherr, John M. Levorse &
Shirley M. Tilghman
Nat Genet. 2003 Jan;33(1):66-9.
Background
• An overall coordination between the expressions of
genes is required for the proper development of an
individual.
• Although most genes are expressed from both the
constituent alleles of the genome, a small subset of
autosomal genes are preferentially expressed from
only one of the parental alleles, a phenomenon known
as genomic imprinting.
• Genomic imprinting relies on establishing and
maintaining the parental-specific methylation of DNA
elements that control the differential expression of
maternal and paternal alleles.
Background, contd.
• The imprinted H19 and Igf2 genes are considered
paradigms of genomic imprinting as their monoallelic
expression pattern is coordinated by a short stretch of
sequence located upstream of H19, known as the
imprinting control region (ICR).
Background, contd.
• This region shows differential methylation, with
•
hypermethylation specifically on the paternal allele.
On the maternal allele this region acts as an insulator
and harbours maternal specific hypersensitive sites.
• The hypersensitive sites were identified as the result
of association of the vertebrate insulator protein CTCF
with the region.
H19 and Igf2
• H19 ( a gene with in vitro growth inhibitory capacity,
and encodes fetal liver mRNA) and Igf2 (encodes
insulin-like growth factor 2) are part of a cluster of
imprinted genes on mouse chromosome 7.
• The genes exhibit reciprocity in allele-specific
expression. Only the maternal allele of H19 is
expressed whereas for Igf2, it is the paternal allele
that is active.
• Expression of the two genes in endodermal tissues is
dependent on a common set of enhancers located
between 7 and 9 kb downstream of the H19 promoter.
H19 and Igf2, contd.
• The imprinting of the two genes is mechanistically
linked. Deletion of H19 and the ~10-kb region
upstream of it leads to biallelic expression of Igf2.
• Sequences upstream of H19 is important for
monoallelic expression of both H19 and Igf2.
CTCF
• The nuclear protein,
CTCF is an evolutionarily
conserved zinc finger
(ZF) phosphoprotein that
binds through
combinatorial use of its
11 ZFs to ~50 bp target
sites that have
remarkable sequence
variation.
CTCF, contd.
• Formation of different CTCF–DNA complexes, some
of which are methylation-sensitive, results in distinct
functions, including gene activation, repression,
silencing and chromatin insulation.
CTCF, contd.
• CTCF is important in the formation of an epigenetically
regulated chromatin insulator at the ICR, which in turn
controls the expression pattern of H19 and Igf2.
• CTCF plays an imprortant role in the maintenance of
the methylation profile of the region.
• Disrupting the spectrum of target specificities by ZF
mutations or by abnormal selective methylation of
targets is associated with cancer.
Hypothesis
• The presence of CTCF in male and female
germ cells indicates that it could participate in
establishing the epigenetic state of the ICR
during gametogenesis.
• CTCF binding may maintain the unmethylated
state of the maternal ICR throughout
development.
Introducing mutant ICR into ES cells
Methods:
Homologous
recombination
Southern blot
Electrophoretic mobility
shift assay
probes: oligos for the
four wt CTCF sites
Competitors: mutant or
wild-type ICR
Fig1. Targeting of the mutant and wild-type ICR and
the relative binding affinity of mutant CTCF sites.
Conclusion: Mutant CTCF sites in the ICR have markedly less CTCF
binding affinity
CTCF binding maintains the unmethylated
state of the maternal ICR
Methods:
Southern blot
Genomic DNA from
neonatal liver was digested
by restriction enzymes.
DNA fragments was
probed with a 2.4-kb ICR
fragment.
Fig2. Methylation analysis of the mutant ICR
CTCF binding is not necessary for preventing
ICR from being methylated during oogenesis
and early stage of embryo development
Methods:
Bisulfite sequencing
A commonly used technique
for detecting the methylation
pattern of target sequence.
Fig3. Bisulfite sequencing of regions of
maternally transmitted mutant ICRs.
Mutation of ICR, which give rise to low CTCF
binding, leads to expression of Igf2 on the
mutant marternal allele
Methods:
RNase protection
assay
Fig4. Allelic Igf2 mRNA expression in neonatal mice.
Mutation of ICR, which give rise to low CTCF
binding, results in the reduction of maternal
H19 expression
Fig5. Effect of the mutant ICR on maternal H19 expression and promoter
methylation
Overall Conclusions
•• The
CTCF
presence
binding
may
CTCF
maintain
in male
and
unmethylated
female
CTCF
binding of
is necessary
tothe
maintain,
but not
state
germ
of
cells
thethe
indicates
maternal
that
ICRitmethylation
throughout
could participate
in
establish,
differential
of the ICR.
establishing the epigenetic state of the ICR
development.
during gametogenesis.
• mutant ICR lacks enhancer-blocking activity, as
the expression of Igf2 is activated on mutant
maternal chromosomes.
• CTCF binding is necessary for full expression of
maternal H19.
• The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted,
resulting in silencing of the maternal and paternal alleles, respectively.
This event is dependent upon an imprinted-control region two kilobases
upstream of H19. On the paternal chromosome this element is
methylated and required for the silencing of H19. On the maternal
chromosome the region is unmethylated and required for silencing of
the Igf2 gene 90 kilobases upstream. We have proposed that the
unmethylated imprinted-control region acts as a chromatin boundary
that blocks the interaction of Igf2 with enhancers that lie 3' of H19. This
enhancer-blocking activity would then be lost when the region was
methylated, thereby allowing expression of Igf2 paternally. Here we
show, using transgenic mice and tissue culture, that the unmethylated
imprinted-control regions from mouse and human H19 exhibit enhancerblocking activity. Furthermore, we show that CTCF, a zinc finger protein
implicated in vertebrate boundary function, binds to several sites in the
unmethylated imprinted-control region that are essential for enhancer
blocking. Consistent with our model, CTCF binding is abolished by DNA
methylation. This is the first example, to our knowledge, of a regulated
chromatin boundary in vertebrates.
• Chromatin boundaries create independent regions of
•
•
gene expression by preventing the expansion of
heterochromatin and by blocking enhancers from
activating inappropriate promoters. An element with
enhancer-blocking activity must reside between a
promoter and an enhancer to exert its repressive effect.
the ICR contains two maternal-specific nuclease
hypersensitive regions: HS1, which maps between -4.1
and -3.6 kilobases (kb), and HS2, between -2.7 to 2.1 kb upstream of H19. Both are candidate sites for
boundary protein binding.
the H19 ICR has enhancer-blocking activity and is not a
silencer.
• both hypersensitive regions of the ICR are
required for full enhancer-blocking activity in
vivo. In contrast, the sequence between HS1
and HS2 (IVS) had minimal blocking activity.
• A CpG-rich 45-bp sequence in the human B
repeats was found to be 60% identical to two
sites in HS1 and three sites in HS2, as reported.
CTCF, a zinc-finger protein implicated in both
transcriptional activation and repression, is
required for the enhancer-blocking function of
several vertebrate boundary or insulator
elements.
• Maternal chromosome-specific hypersensitivity to
•
nuclease digestion has been demonstrated at two
regions that are ~ 2.4 kb and 3.8 kb upstream of the
H19 promoter. Also, this region displays paternal
chromosome-specific hypermethylation that extends
from approximately 4.0 kb to 2.0 kb. Therfore,
methylation of this region has been suggested to be
responsible for controlling the imprinted expression of
H19 and Igf2.
A 2-kb region located 5' to the imprinted mouse H19
gene is hypermethylated on the inactive paternal allele
throughout development.
• The mouse H19 gene encodes one of the most abundant RNAs
in the developing mouse embryo. It is expressed at the
blastocyst stage of development, and accumulates to high
levels in tissues of endodermal and mesodermal origin. After
birth the gene is expressed in all tissues except skeletal muscle.
It lacks a common open reading frame in the 2.5-kilobase RNA,
but has considerable nucleotide sequence similarity between
the genes of rodents and humans. Expression of the gene in
transgenic mice results in late prenatal lethality, suggesting that
the dosage of its gene product is strictly controlled.
• Nature. 1995 May 4;375(6526):34-9.
Disruption of imprinting caused by deletion of the H19 gene region
in mice.
Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman
SM.
The imprinted H19 gene, which encodes an untranslated RNA, lies at the
end of a cluster of imprinted genes in the mouse. Imprinting of the insulin-2
and insulin-like growth factor 2 genes, which lie about 100 kilobases
upstream of H19, can be disrupted by maternal inheritance of a targeted
deletion of the H19 gene and its flanking sequence. Animals inheriting the
H19 mutation from their mothers are 27% heavier than those inheriting it
from their fathers. Paternal inheritance of the disruption has no effect,
which presumably reflects the normally silent state of the paternal gene.
The somatic overgrowth of heterozygotes for the maternal deletion is
attributed to a gain of function of insulin-like growth factor 2, rather than a
loss of function of H19.
• A role for CTCF-mediated enhancer blocking activity has been demonstrated
most clearly at the Igf2/H19 locus in mouse and human (10-12). In this
imprinted locus the maternally transmitted allele expresses H19 but not Igf2,
whereas the paternally transmitted allele expresses Igf2 but not H19.
Furthermore the paternal allele is methylated at a site (the ICR or imprinted
control region) located between the two genes (Fig. 5). Earlier work had
suggested that the ICR might contain an enhancer blocking activity that
would prevent downstream endodermal enhancers from activating Igf2.
Direct examination reveals that the ICR contains four CTCF binding sites in
the mouse ICR and seven in human. Methylation of these sites abolishes
CTCF binding. These results indicate that CTCF plays an important role as an
insulator protein in allele-specific regulation at this imprinted locus, and that
the insulator function can be modulated by DNA methylation, thus making
the CTCF sites susceptible to epigenetic regulation. Quite recently a cluster
of differentially methylated CTCF sites has been identified at the Xist gene
promoter, and it has been suggested that these are enhancer-blocking
elements important for X chromosome inactivation (13).
Background
Certain loci in the mammalian genome exhibit
functional inequivalence of the two alleles.
Depending on the parent of origin, some genes
are expressed exclusively from the maternal
chromosome and others exclusively from the
paternal chromosome.
Genomic imprinting relies on establishing and
maintaining the parental-specific methylation of
DNA elements that control the differential
expression of maternal and paternal alleles.
• Strong support in favor of this role also
comes from mutant studies in which DNA
methyltransferase gene (dnmt) has been
deleted. In these mutants, H19 was
shown to be expressed in a biallelic
manner, whereas Igf2 expression was
completely lost (Li et al. 1993 ).