A DNA-binding protein, e.g., transcription factor or repressor, sensitive to DNA méthylation, has been implicated in the regulation of imprinted Xist expression in preimplantation embryos in that the expressed paternal allele is unmethylated whereas the repressed maternal allele is methylated at specific CpG sites within the promoter region (Zuccotti and Monk, 1995). Theoretically, there are two possible modes of repressive action by DNA méthylation (Fig. 4.12):
(1) transcription factor(s) itself is methylation-sensitive, i.e., the transcription factor(s) cannot bind to the methylated sequence (activator model. Fig. 4.12A) or
(2) a repressor protein binds to the methylated sequence and inhibits binding of the transcription factor(s) (repressor model. Fig. 4.12B).
Whichever the case may be, the end result is the cessation of transcription (in my research, of the luciferase gene).
Recently Huntriss et a l (1997) within the Molecular Embryology Unit at ICH have identified a protein in ES cells that binds to a methylated Xist promoter
sequence, 5'-GCGCCGCGG-3' (nt -44 to -36), and have shown that this sequence, in its unmethylated form, is important for transcription. Their results in ES cells
encouraged me to favour the repressor model (Fig. 4.12B) as a working hypothesis and to search for a similar (or the same) protein in preimplantation embryos. If such a
Figure 4.11
Summary of the effect of in vitro méthylation on activity of the pXist-lucI construct. Open rectangles indicate the 233 bp Xist promoter fragment (-220 to +13). Hooked arrows indicate the transcription start site. Vertical lines on the open rectangles show CpG sites methylated within the promoter; no site for HpaU methylase,three sites for FnuDll methylase, two sites for Hhal methylase, five sites for FnuDU+Hhal methylases, and 12 sites for Sssl methylase. Total numbers of CpG sites methylated in the whole construct are given in parentheses.
Luciferase activity in 2-cell embryos is shown as mean (black bar) ± standard error of the mean (horizontal line). In both Experiments 1 and 2, the decreases in luciferase activity seen with 2-cell embryos microinjected with Hhal+FnuDll- methylated and tel-m ethylated constructs are statistically significant, compared to the luciferase activity in 2-cell embryos microinjected with the unmethylated construct. For the details of each Experiment, refer to Figures 4.9 and 4.10 and Tables 4.2 and 4.3.
Experiment 1
CpG sites Methylase Luciferase activity
1- Unmeth h Hpall (24) h FnuDII (20) -T h Hhal (28) I- Fn+Hh (43) Sssl (308) 50 100 150 200 250 300 Luciferase (RLU x 10 ^)
Experiment 2
CpG sites M ethylase-L
-i
11 -c J - Unmeth > Hpall (24) ] - FnuDII (20) > Hhal (28) H — ~l- Fn+Hh (43) Sssl (308) Luciferase activity 5 10 15 Luciferase (RLU x 10^)Figure 4.12
Models for repression of a methylated maternal allele of the Xist gene in
preimplantation embryos. Solid horizontal lines indicate the Xist promoter region. Closed rectangles indicate the 5' end of the first exon. Five methylation-sensitive restriction enzyme sites are indicated by vertical lines and these sites correspond to, from 5' upstream to 3' downstream, Hhal, Mlul, SnaBl, Hhal and SacB sites in Figure 4.1. Open circles denote unmethylation. Closed circles denote
méthylation. Hooked arrows indicate transcription (and also the transcription start site).
(A) Activator model. An activator (transcription factor; T) binds to a sequence encompassing the differentially methylated CpG sites within the promoter only when the sequence is not methylated. In this model, the activator itself is methylation-sensitive and no other sequence-specific factor is involved. (B) Repressor model. A repressor (R) specifically binds to the methylated sequence and mediates the repressive effect of DNA méthylation by competing with the transcription factors. In this model, the transcription factor can be either methylation-sensitive or methylation-insensitive.
paternal allele maternal allele
©
(D
g
B
paternal allele maternal alleleprotein were active in repressing a methylated Xist promoter inherited from the oocyte, then co-injection with a methylated oligonucleotide encompassing the differentially methylated Xist promoter sequence might be expected to compete for the protein ('mop up') and allow increased expression from the methylated pXist-lucI construct (Fig. 4.13).
To test whether the methylation-mediated repression of the pXist-lucI construct is alleviated by co-injection with the methylated oligonucleotide, 5551- methylated pXist-lucI (fixed at 25 ng/pl) was co-injected {in vivo competition) with different concentrations of the 555Ï-methylated 22 bp oligonucleotide,
5'-GCCTTCAGCGCCGCGGATCAGT-3' (nt -51 to -30; see also Figure 4.1C). This 22 bp oligonucleotide contains the three CpG sites, -44 to -36, which have been reported to be differentially methylated in eggs and sperm (Zuccotti and Monk, 1995) and recognised by a methylation-dependent DNA binding protein in ES cells
(Huntriss et a i, 1997). Méthylation of the 22 bp oligonucleotide was confirmed by digestion of the 555l-methylated product with methylation-sensitive restriction enzymes, Hhal, Acil and Sacll (Fig. 4.14).
The methylated oligonucleotide was co-injected at two different
concentrations, 0.1 and 2 ng/jil (which correspond to an equimolar amount and 20 times molar excess, respectively) of a fixed concentration (25 ng/pl) of the 5.4 kb pXist-lucI construct. Out of 38 embryos surviving microinjection, 30 (78.9%) cleaved to 2-cell embryos and 8 (21.1%) were arrested at the 1-cell stage. Luciferase activities of individual embryos are plotted in Figure 4.15. Embryos injected with the 555l-methylated pXist-lucI construct alone showed a luciferase activity of 638 ± 184 RLU, only 2% of the level of luciferase activity seen for the unmethylated pXist-lucl. When the 555l-methylated pXist-lucl was co-injected with 0.1 and 2 ng/pl Sssl- methylated oligonucleotide, average luciferase activity did indeed increase, in a dose-
Figure 4.13
The principle of in vivo competition assay. The principle is based on the repressor model shown in Figure 4.12B. The repressor protein binds to the sequence
encompassing the three methylated CpG sites (-44 to -36; within the recognition sequences of Hhal and Sacll) and inhibits binding of the transcription factor to that site.
(A) Co-injection of the methylated pXist-lucl construct and methylated 22 bp oligonucleotide (-51 to -30). The co-injected 22 bp oligonucleotide competes for the repressor protein and allows the binding of the transcription factor to the methylated Xist promoter sequence in the pXist-lucI construct, resulting in transcription of the luciferase gene.
(B) Co-injection of the methylated pXist-lucI and unmethylated 22 bp oligonucleotide. The unmethylated oligonucleotide does not compete for the repressor and, therefore, transcription does not occur. Vertical lines indicate the methylation-sensitive restriction enzyme sites, corresponding to those in Figures 4.1 and 4.12. Closed circles indicate méthylation. Open circles indicate unmethylation.
-51 m m m -30 5 ’-G CCTTCA G CG CCG CG G A TCA G T-3’
. . .
H
luciferase gene
<î>
H luclluciferase gene
B
5 ’-G C C TTCA G CGCCG CGG A TCA G T-3’
00
fit
H luciluciferase gene
00
fit
Figure 4.14
Confirmation of méthylation of the 22 bp oligonucleotide. The 22 bp oligonucleotide (-51 to -30) was methylated in vitro with Sssl methylase. (A) Sites of CpG méthylation recognized by methylation-sensitive restriction enzymes, Hhal, Acil and SacU. All three CpG sites are methylated with Sssl methylase and méthylation of each site can be confirmed by digestion with a relevant restriction enzyme. The recognition sequences of the enzymes are 5 -GCGC-3' for Hhal, 5 -CCGC-3' for Ac/I and 5 -CCGCGG-3' for SacU.
The 5' termini of the oligonucleotide were labelled by y-32p dATP (® ) with the polynucleotide kinase for autoradiogram following electrophoresis.
(B) Results of autoradiogram. The digested oligonucleotides (methylated and mock-methylated) were fractionated on a 6% polyacrylamide gel. The position of the original (uncut) 22 bp oligonucleotide kinase is shown by the arrow. Lower bands indicate digested smaller fragments. Although restriction enzyme digestion is not complete (as seen by the remaining 22 bp bands in mock-methylated lanes), there is a clear difference in sensitivity (resistance) to enzyme digestion between methylated and mock-methylated oligonucleotides, confirming that most
molecules of the ^jjl-methylated oligonucleotide are methylated at the three CpG sites.
CO = ü T < œ
i l l
m m m © 5 ' -GCCTTCAGCGCCGCGGATCAGT-3 3 ' -CGGAAGTCGCGGCGCCTAGTCA-5B
M ethylated oligo 1 f M ock-methylated oligo 1 Ü Ü CO — o CO c - C Ü CO CO u X < C/) ■22 bpFigure 4.15
Results of in vivo competition assay 1. The tol-m ethylated pXist-lucI was co injected with the 555l-methylated 22 bp oligonucleotide (-51 to -30) into the male pronucleus of 1-cell embryos. The DNA constructs were premixed before
microinjection. The concentration of the pXist-lucI was fixed at 25 ng/|il. The oligonucleotide was co-injected at two different concentrations, 0.1 ng/|xl and 2 ng/|il, which correspond to the equimolar and 20-fold molar excess of the pXist-lucI, respectively. Lucifease activity is plotted on a log scale. Open circles indicate 2-cell embryos and open squares indicate arrested 1-cell embryos. Mean (closed circles) and standard error of the mean (vertical lines) for the 2-cell embryos are also shown. The increases in luciferase activity seen with 2-cell embryos co-injected with the methylated oligonucleotide are not statistically significant, compared to the luciferase activity in 2-cell embryos injected with the tol-m ethylated pXist-lucI alone (see Table 4.4).
100000 n
10000
-CO
1000
:100 -I
pXist-lucI me(-) me(+) me(+) me(+)
e m b r y o s (RLU, m e a n ± s . e . m . ) p X i s t - l u c I o l i g o (ng/|Lil) t r a t i o P v a l u e me ( - ) 0 2 - c e l l 8 2 7 9 9 2 ± 6 43 1 a r r e s t e d 1 - c e l l 1 507 me ( + ) 0 2 - c e l l 6 638 ± 184 3 . 6 4 P < 0 . 0 0 5 ^ a r r e s t e d 1 - c e l l 1 11 76 me ( + ) 0 . 1 2 - c e l l 5 806 ± 288 - 0 . 1 5 0 . 5 0 < p b a r r e s t e d 1 - c e l l 1 8914 me ( + ) 2 2 - c e l l 11 1 8 0 8 ± 766 - 1 . 1 0 0 . 2 0 < P < 0 . 5 0 ^ a r r e s t e d 1 - c e l l 5 3 2 1 7 3 ± 1 55 07 L u c i f e r a s e ( l u e . ) a c t i v i t y i s p r e s e n t e d a s m ean ± s t a n d a r d e r r o r o f t h e m e an ( s . e . m . ) . RLU, r e l a t i v e l i g h t u n i t s . F o r s t a t i s t i c a l a n a l y s i s , S t u d e n t ' s t - t e s t w a s p e r f o r m e d . F o r t h e d e t a i l s o f t h e c a l c u l a t i o n o f t h e t r a t i o a n d P v a l u e , s e e t h e l e g e n d t o T a b l e 4 . 2 .
l u e . a c t i v i t y w a s c o m p a r e d w i t h t h a t o f t h e 2 - c e l l e m b r y o s i n j e c t e d w i t h t h e m e t h y l a t e d p X i s t - l u c I c o n s t r u c t a l o n e .
00
dependent manner, to 806 ± 288 RLU and 1,808 ± 766 RLU, respectively, although these increases were not significant (Table 4.4). It should be noted that a higher proportion of embryos were arrested at the 1-cell stage following co-injection with the 2 ng/|il oligonucleotide than with the 0.1 ng/|il of oligonucleotide, and that the
arrested embryos co-injected with the 2 ng/|il oligonucleotide showed a significantly high luciferase activity.
To see whether the increase in luciferase activity in the 2-cell embryos following co-injection with the 2 ng/p.1 tol-m ethylated oligonucleotide is
methylation-specific, luciferase activity was compared following co-injection of the 5M-methylated pXist-lucI (25 ng/p,l) with the 2 ng/|jl of either unmethylated or t o l - methylated oligonucleotide. Out of 35 embryos surviving microinjection, 31 (88.6%) cleaved to the 2-cell stage, three were arrested at the 1-cell stage and one (2.9%) died. One embryo was lost during collection. Luciferase activity is plotted in Figure 4.16. Injection of the 5jjl-methylated pXist-lucI construct alone gave an average luciferase activity of 171 ± 77 RLU, only 4% of the level of luciferase seen with the
unmethylated pXist-luc 1 (although not statistically significant due to a small sample size; see Table 4.5). When the tol-m ethylated pXist-lucI was co-injected with t o l - methylated oligonucleotide, luciferase activity increased slightly to 627 ± 1 4 4 RLU, but the increase was not significant, compared to the luciferase activity in embryos microinjected with the tol-m ethylated pXist-lucI alone (0.10cP<0.20). However, a similar extent of increase in luciferase activity, 532 ±221 RLU (0.20cP<0.50, compared to the luciferase activity seen with the 5jjl-methylated pXist-lucI alone), was also observed following co-injection with the unmethylated oligonucleotide. The difference in luciferase activity between embryos microinjected with the Sssl-
methylated pXist-lucI and tol-m ethylated oligonucleotide and those with the Sssl- methylated pXist-lucI and unmethylated oligonucleotide was also not significant (t = -0.35, 0.50<P) in this assay.
Figure 4.16
Results of in vivo competition assay 2. The 555l-methylated pXist-lucI was co- injected with unmethylated or tol-m ethylated 22 bp oligonucleotide (-51 to -30) into the male pronucleus of 1-cell embryos. The DNA constructs were premixed before microinjection. The pXist-lucI and the oligonucleotide were injected at concentrations of 25 ng/|il and 2 ng/|xl, respectively. These concentrations give 20-fold molar excess of the oligonucleotide to the pXist-lucl. Luciferase activity is plotted on a log scale. In order to plot a zero measurement on the log scale, all data (X) are plotted as X+1. Open circles indicate 2-cell embryos and open squares indicate arrested 1-cell embryos. Mean (closed circles) and standard error of the mean (vertical lines) for the 2-cell embryos are also shown. Compared to the luciferase activity in 2-cell embryos microinjected with methylated pXist-lucl alone, a slight, but not significant, increase in luciferase activity was observed in 2-cell embryos following co-injection with the tol-m ethylated oligonucleotide. However, a similar extent of increase was also observed following co-injection with the unmethylated oligonucleotide. For statistics, see Table 4.5.
10000
b 1000
100
<4 -1 me(+) me(+)pXist-lucl me(-) me(+)
constructs iniected stage N o . embryos
lue. activity (RLU, mean+s.e.m.)
statistics
pXist-lucl oligo t ratio P value
me (-) none 2-cell 4 3943 ± 1617 arrested 1-cell 0 me ( + ) none 2-cell 3 171 ± 77 1.97 0.10<P<0.20^ arrested 1-cell 0 me ( + ) me ( + ) 2-cell 11 627 ± 144 -1.59 0.10<P<0.20^ arrested 1-cell 0 me ( + ) me (-) 2-cell 13 532 ± 221 -0.76 0.20<P<0.5Qb arrested 1-cell 3 714 ± 324
Luciferase (lue.) activity is presented as mean ± standard error of the mean (s.e.m.). RLU, relative light units.
For statistical analysis, Student's t-test was performed. For the details of the calculation of
pXist-lucl construct alone.
4.4 DISCUSSION
In female mouse preimplantation embryos, Xist expression is imprinted; the paternal allele is expressed (albeit at low level) and the maternal allele is repressed (Kay gf aA, 1993, 1994; Latham gr a/., 1994). This imprinted Xwr expression precedes XCI, so that preferential expression of the paternal Xist allele in preimplantation embryos is correlated with preferential paternal XCI in the extraembryonic tissues. This correlation does not necessarily mean that early Xist transcription from the paternal X chromosome in preimplantation embryos is the cause of imprinted paternal XCI at the blastocyst stage. However, the requirement of Xist in cis for preferential paternal XCI in the extraembryonic tissues as documented in Xw?-deficient mice (Marahrens et a l, 1997), and the ability of a Y AC transgene, containing the Xist gene and 9 kb of 5'- and 6 kb of 3'-flanking regions, to initiate inactivation of the autosomal region surrounding the integration site in differentiating ES cells (Herzing et a l, 1997), strongly suggest that the Xist gene ox Xist RNA plays an important role in preferential paternal XCI.
Imprinted Xist expression is only observed in preimplantation embryos and in the first delineating extraembryonic tissues, but this preferential expression of the paternal allele is not perpetuated into the soma. In this respect, Xist is not a typical imprinted gene. However, Xist provides an excellent model to investigate gametic imprinting with effect immediately after fertilisation and erased during embryogenesis so that the imprint is not observed in somatic cells. DNA méthylation is a strong candidate for gametic imprinting and the control of Xist expression (Zuccotti and Monk, 1995; Ariel et a l, 1995). The correlation between the erasure of the
imprinting mark for Xist expression by the time of gastrulation and the genome-wide déméthylation by the blastcyst stage further supports the idea of control of early Xist expression by DNA méthylation (see below).
Although knock-out experiments and transgenic experiments have established the significance of Xist in XCI (for details, see Tables 1.4 and 1.5 in Chapter 1), little is known about the regulatory role of the Xist promoter itself and the molecular mechanisms involved in imprinted expression in preimplantation embryos. To date, only a few studies have been reported on functional analyses of the Xist promoter (Fillet et ah, 1995; Komura e ta l, 1997; Huntriss e ta l, 1997).
Fillet et al. (1995) have reported that a short fragment of the Xist promoter region, -81 to 4-1, is sufficient to drive expression of a reporter gene in a transient transfection assay in murine cultured cells. They showed that the transgene Xist promoter (up to -1,157 bp upstream from the transcription start site) is active to a similar extent in both Xwr-expressing XX cells and Xwr-negative XY cells. Their results suggest that the repression of the endogenous Xist in male XY cells is not due to the absence of transcription factors required for Xist expression but due to the presence of a cis- and/or rran^-acting regulatory mechanism to inactivate the Xist gene. They also showed, in an electrophoretic mobility shift assay, that TATA box- binding protein (TBF) binds to the sequence, 5 -TTAAAG-3' (-30 to -25) in the promoter sequence.
Komura et al. (1997), using an in vivo footprinting assay in cultured somatic cells, have identified footprints with consensus seuqences for transcription factors; CCAAT box (-115 to -101), two Spl sites (-73 to -65 and -50 to -46) and TATA box (-30 to -25). However, the implicated protein binding was not substantiated in in vitro assay in their study.
Huntriss et al. (1997) have shown, by an electrophoretic mobility shift assay and a transient transfection assay, that a protein in ES cells binds to a methylated Xist promoter sequence, 5'-GCGCCGCGG-3' (-44 to -36), and that this sequence is
important for transcription in its unmethylated form (see below). This i s # e strong evidence for control of Xist transcription by DNA méthylation via a repressor protein.
In this Chapter, I have investigated the activity of a 233bp fragment (-220 to +13) of the Xist promoter following microinjection into mouse 1-cell embryos and the involvement of DNA méthylation in its regulation.