11 research outputs found
Complete biallelic insulation at the H19/Igf2 imprinting control region position results in fetal growth retardation and perinatal lethality.
The H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken β-globin insulator (ChβGI)(2) are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChβGI)(2) does not become methylated in fetal male germ cells. The (ChβGI)(2) is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChβGI)(2) sequences from methylation in the male germ line.We genetically dissected the ChβGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChβGI)(2) significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChβGI)(2) from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChβGI)(2) lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission.In this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development
Paternal transmission results in late fetal/neonatal lethality phenotype.
<p>Normal (+/+) outbred CF1 and inbred, 129S1 and FVB, mothers were crossed with m(ChβGI)<sub>2</sub>/+ fathers and the offspring was genotyped at weaning. The number of wild type +/+ and mutant +/−(P) heterozygous young from each litter is given per row. Numbers in parentheses</p>†<p>indicate dead pups of greatly reduced size, found on the day of birth.</p
DNA methylation of the (mChβGI)2 in 18.5 dpc fetuses.
<p>(A) Bisulfite sequencing was performed to analyze CpG methylation of the (mChβGI)<sub>2</sub> using genomic DNA from 18.5 dpc fetuses. Genotypes are indicated on top. Maternal (M) or paternal (P) transmission of the allele is indicated on the right. Unmethylated CpGs (white squares) and methylated CpGs (black squares) are shown along independent chromosomes (horizontal lines). Two siblings were assessed in each case, separated by space between groups of chromosomes. Simple arrow indicates the CTCF site. Double arrows and asterisk indicate the positions of the USF1 and VEZF1 deletions. (B) Southern blot hybridization results in kidneys (K) and livers (L) after paternal and maternal transmission. The (ChβGI) sequence was used as a probe. The two diagnostic <i>Hpa</i>II/<i>Msp</i>I sites and the <i>Bam</i>HI and <i>Bgl</i>II restriction sites are indicated. (C) Bisulfite sequencing of the ICR sequences from the same samples as in (A).</p
Role of CTCF Binding Sites in the Igf2/H19 Imprinting Control Region
A ∼2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis—the H19 promoter and gene—also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR
Targeting the (mChβGI)2 to the <i>H19/Igf2</i> ICR.
<p>The ICR was replaced by the (mChβGI)<sub>2.</sub> The CTCF sites were in the same orientation as the endogenous CTCF sites in the ICR. Novel restriction sites, such as <i>Stu</i>I were generated at the sites of the binding site deletions. One control +/+ and four +/− ES cell clones are shown out of 24 that underwent conservative recombination. The <i>lox</i>P sites-flanked <i>neo</i> selection cassette was removed by <i>Cre</i>-mediated excision.</p
DNA methylation of the (mChβGI)2 in 18.5 dpc fetal germ cells.
<p>Bisulfite sequencing results are shown from +/−(P) fetuses. (A) The paternally inherited (mChβGI)<sub>2</sub> allele in female germ cells. (B) The paternally inherited (mChβGI)<sub>2</sub> allele in male germ cells. (C) The maternally inherited ICR sequences in male germ cells are shown as controls. The percentage of methylated CpGs is indicated for each allele. The bar above indicates the position of the previously analyzed CpGs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012630#pone.0012630-Szab3" target="_blank">[44]</a> with the % of methylated CpGs in this subset. Chromosomes from independent bisulfite reactions are grouped. Other details are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012630#pone-0012630-g004" target="_blank">Figure 4</a>.</p
Phenotype of 18.5 dpc fetuses inheriting the (mChβGI)2.
<p>(A) Representative +/+ and +/–(P) fetuses are shown. (B) Expression of <i>Igf2</i> and <i>H19</i> was measured by real-time RT-PCR in kidneys. RNA from two +/–(P) and –(M)/+ fetuses (samples 3–4 and 7–8, respectively) and their +/+ littermates (samples 1–2 and 5–6) was analyzed. (C) Allele-specific expression of <i>H19</i> in kidneys and livers of the same fetuses was measured by RT-PCR SNuPE. The % expression of the maternal (M) and paternal (P) allele in the total expression is shown.</p
Weight of 18.5 dpc fetuses on maternal and paternal inheritance of the (mChβGI)<sub>2.</sub>
<p>∧ Sibling from -/(M) ♀ X +/+ ♂ matings.</p>‡<p>Sibling from +/+ ♀ X +/−(P) ♂ matings.</p>*<p>Sibling from +/+ ♀ X +/−(P chimera #2) ♂ matings.</p>†<p>Sibling from +/+ ♀ X +/−(P) ♂ matings. +/+ females were from transgenic line TgOG2.</p><p>¶ <i>P</i><0.0001.</p><p>$ <i>P</i><0.025.</p><p>(M), Maternal allele; (P), Paternal allele.</p
Lethality is caused by strict biallelic insulation at the IC1 in mouse chromosome 7.
<p>(A) Maternal (pink) duplication of distal chromosome 7 (MatDup.dist7) fetuses carry biallelic insulation (STOP signal) at the imprinting control center 1 (IC1) and die. The lethality phenotype is rescued by maternal transmission of one copy of the mutant IC1 that lacks CTCF binding (x) and insulator function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012630#pone.0012630-Han2" target="_blank">[81]</a>. The imprinting control center 2 (IC2) is bi-maternal. Correction of biallelic ICR insulation to monoallelic insulation is sufficient to rescue perinatal lethality of the MatDup.dist7 genotype. (B) Our present experiments provide the reciprocal argument: introducing strict biallelic insulation to the IC1 causes lethality. By substituting the paternal chromosome's (light blue) methylated (black lollipop) ICR with the unmethylated (ChβGI)<sub>2 </sub><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012630#pone.0012630-Szab3" target="_blank">[44]</a> or the (mChβGI)<sub>2</sub> we introduced biallelic insulation into the IC1 and IC2 remained intact. Lethality was observed in the +/(mChβGI)<sub>2</sub> but not in the +/(ChβGI)<sub>2</sub> genotype 44]. By removing the USF and VEZF1 binding sites from the (ChβGI)<sub>2</sub>, biallelic insulation has become complete, causing death in the +/(mChβGI)<sub>2</sub> genotype.</p