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Fig. 6. Differentiating ES cells mimic the developmental gain of
Igf2r/Airn imprinted expression. Changes in
Igf2r/Airn expression (black lollipop, no expression; dotted arrow,
low-level expression; solid arrow, medium- or high-level expression), DNA
methylation (Me), repressive histone modifications (black; K9, H3K9me3; K27,
H3K27me3), active histone modifications (white; K4, H3K4me2/me3; Ac, H3K9Ac),
shown on the left-hand side for the mouse embryo and on the right-hand side
for ES cells. In pre-implantation embryos, Igf2r is biallelically
expressed and Airn is silent. The histone modification status is
unknown, but DNA methylation is present on the maternal Airn CpG
island and is absent from the Igf2r CpG island. These patterns are
mimicked in undifferentiated ES cells that also show repressive H3K9me3
modifications on the maternal Airn CpG island and bivalent histone
modifications comprising H3K27me3 and H3K4me3 on the Igf2r and
Airn promoters, which is typical of all CpG islands in ES cells
irrespective of expression status. In 11.5-13.5 dpc embryos, Igf2r
shows imprinted maternal-specific expression, whereas Airn shows
imprinted paternal-specific expression in all tissues except post-mitotic
neurons (which lack Airn and express Igf2r biallelically).
Persistent expression of the paternal Igf2r promoter is detected in
some tissues of the post-implantation embryo. DNA methylation is maintained on
the maternal Airn promoter and is also now present on the paternal
Igf2r promoter (the latter is not fully methylated until after
birth). Active histone marks (H3K4me2/3, H3K9Ac) are only found on the
expressed paternal Airn and expressed maternal Igf2r CpG
island promoters. Repressive histone marks (H3K9me3, H4K20me3) plus HP1 are
only found on the silent maternal Airn and the silent paternal
Igf2r promoters. Notably, both the expressed and silent Airn
and Igf2r promoters and their gene bodies are free of H3K27me3. All
these features, including the persistent low-level expression of the paternal
Igf2r promoter and the loss of H3K27me3, are fully mimicked in
differentiated ES cells. We propose two models, as discussed in the text, to
explain the persistence of low-level Igf2r expression from the
paternal allele: (1) maternal-specific upregulation and (2) paternal-specific
silencing with stochastic `escapers'. References:
1(Szabo and Mann,
1995 ), 2(Lerchner
and Barlow, 1997),
3(Wang et al.,
1994), 4(Terranova
et al., 2008),
5(Stoger et al.,
1993), 6(Braidotti
et al., 2004),
7(Mikkelsen et al.,
2007), 8(Sleutels
et al., 2002),
9(Regha et al.,
2007). M, maternal; P, paternal;?, unknown status; d RA, days of
RA-induced differentiation.
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50:1). (B)
Allele-specific QPCR showing the ratio of maternal to paternal Igf2r
expression during RA-induced differentiation of maternally targeted (S12/+,
dark grey bars) and paternally targeted (+/S12, light grey bars) ES cells.
Mean values and s.d. of three replicates are shown. Since Igf2r is
biallelically expressed in undifferentiated ES cells, the middle value of the
three replicates on day (d) 1 is set to 1 (*). The maternal to
paternal Igf2r expression ratio increases during differentiation in
both S12/+ and +/S12 ES cells, showing that expression of the maternal allele
is greater than that of the paternal allele. (C)Igf2r
expression in feeder-depleted differentiating D3 ES cells with disruption of
full-length Igf2r by lacZ-polyA insertion into exon 1 on the
maternal (lacZ/+) or paternal (+/lacZ) or on both
(lacZ/lacZ) alleles, analysed by northern blot using a
downstream Igf2r probe (HX). Igf2r expression is upregulated
from the wild-type maternal allele (+/lacZ) and is expressed at
similar levels throughout differentiation from the wild-type paternal allele
(lacZ/+). Igf2r expression in lacZ/lacZ ES
cells indicates expression from irradiated feeder MEFs that contaminate
earlier time points, despite feeder depletion. Oct4, control for ES
cell differentiation; 18S rRNA, loading control. (D) Northern blot
confirming biallelic Igf2r expression in undifferentiated
+/lacZ and lacZ/+ D3 ES cells and the absence of
Igf2r expression in lacZ/lacZ D3 ES cells (details
as C). Wild-type D3 and CCE undifferentiated ES cells are shown for
comparison. All ES cells were grown on mutant MEF feeders that completely lack
Igf2r as they have a targeted deletion of the maternal Igf2r
promoter and a Thp (hairpin-tail) deletion on the paternal chromosome that
includes the whole Igf2r imprinted cluster. (E) QPCR analysis
(assay Igf2rex48) of maternal and paternal wild-type Igf2r alleles in
+/lacZ (light grey bars) and lacZ/+ (dark grey bars)
differentiating ES cells grown as in D on mutant feeders. Bars indicate mean
values with s.d. of three replicates. The middle value of the three replicates
on day 1 was set to 1 (*). Expression of the wild-type maternal
Igf2r allele increases during ES cell differentiation from day 2 of
RA treatment onward, whereas expression of the wild-type paternal allele is
constant during early time points (days 1-4) and increases slightly by day
5.
