Question

17-3. Describe in words the role of CTCF plays in the H19/Igf2 mouse gene locus in details. [Hints: paternally-inherited locus, maternally-inherited locus, differentially methylated regions (DMRs) DMR1, DMR2, and imprinting control regions (ICR)]

Answer 1

CTCF (CCCTC-binding factor)-mediated insulation at the H19-Insulin-like growth factor 2 (Igf2)imprinted domain is a classic example for imprinted gene regulation. DNA methylation difference in the imprinting control region (ICR) is inherited from the gametes and subsequently determines parental allele-specific enhancer blocking and imprinted expression in the soma. The CTCF binding in the soma protects the maternal allele from de novo DNA methylation. CTCF binding is not involved in the establishment of the gametic marks at the ICR, but it slightly delays de novo methylation in the maternally inherited ICR allele in prospermatogonia. Between these two genes lies a 2.4 kb long differentially methylated region (DMR) that is required for the monoallelic expression of both the H19 and Igf2 genes, and therefore is called an ICR. Its deletion from the maternal allele results in biallelic Igf2 expression and from the paternal allele in biallelic H19 expression. Methylation of this DMR is exclusive to the paternally inherited chromosome and originates from the sperm . Igf2 expression is also regulated by two additional paternally methylated DMRs. Igf2 DMR1, upstream of the Igf2 gene functions as a mesodermal silencer in the maternal allele while DMR2, in the sixth exon, functions as an enhancer in the paternal allele

CTCF is the master organizer of the maternal allele's chromatin Utilizing single nucleotide polymorphisms (SNPs) between parental mouse lines and using quantitative allele-specific chromatin immunoprecipitation single nucleotide primer extension (SNuPE) assays, we measured the chromatin composition along the H19/Igf2 imprinted domain in normal cells and cells with engineered mutations at the four ICR-CTCF binding sites.

(A) Imprinted insulation at the H19/Igf2 imprinted domain by CTCF binding in the ICR based on publications referenced in the text. Maternal chromosome (M): unmethylated (white lollipops) ICR (shaded area) is inherited from the oocyte. CTCF imparts insulator activity (bracket) between the Igf2 promoters and the shared, downstream enhancers . Initiation of H19 expression depends on an unmethylated ICR during embryogenesis. Paternal chromosome (P): methylated (black lollipops) ICR is inherited from the sperm, CTCF cannot bind, hence ICR has no insulator activity thus,

(B) CTCF binding site mutations in the maternal ICR allele disrupt imprinted expression (Szabó et al., 2004). CTCF no longer binds in the mutant maternal chromosome (M_CTCFm), thus, the enhancers can access the Igf2 promoter in both alleles. The mutant ICR is methylated and inactivates the H19promoter.

(C) Non-imprinted insulation at the H19/Igf2 locus by the chicken β-globin insulator duplex (ChβGI)₂ (orange rectangle) (Szabó et al., 2002). The (ChβGI)₂ is unmethylated and insulates the Igf2promoter from the shared enhancers when substituted for the ICR and transmitted maternally (not shown) or paternally (P), with 10% Igf2 activity remaining. H19 is overactivated 1.5-fold by the (ChβGI)₂ sequences in the paternal allele (bold arrow).

(D) Biallelic insulation by the mutant chicken β-globin insulator duplex (mChβGI)₂ (turquoise rectangle) carrying mutations for boundary factor binding sites (stars) (Lee et al., 2010). Insulation is complete, with no detectable remaining Igf2 expression.