CTCF binding is not the epigenetic mark that establishes post-fertilization methylation imprinting in the transgenic H19 ICR

Abstract

Imprinted expression of the mouse Igf2/H19 locus is controlled by parent-of-origin-specific methylation of the imprinting control region (ICR). We previously demonstrated that when placed in a heterologous genomic context, the H19 ICR fragment contains an intrinsic activity that allows it to acquire differential methylation in somatic cells but not in germ cells. In the present study, we investigated the requirements for the CTCF-binding sites of the ICR in the acquisition of post-fertilization methylation. To this end, two mutant ICR fragments were introduced into the human beta-globin locus in a yeast artificial chromosome transgenic mouse (TgM) model: 4xMut had mutations in all four ICR CTCF-binding sites that prevented CTCF binding but retained the methylation target CpG motifs, and -9CG harbored mutations in the CpG motifs within the CTCF-binding sites but each site retained constitutive CTCF-binding activity. In TgM germ cells and pre-implantation blastocysts, the absence of CTCF-binding sites (4xMut) did not lead to hypermethylation of the transgenic H19 ICR. However, after implantation, the mutations of CTCF sites (4xMut and -9CG) affected the maintenance of methylation. These results demonstrated that although the CTCF-binding sites are indispensable for maintenance of the unmethylated state of the maternal ICR in post-implantation embryos, they are not required to establish paternal-allele-specific methylation of the transgenic H19 ICR in pre-implantation embryos.

De novoDNA methylation through 5'-segment of theH19ICR maintains its imprint during early embryogenesis

“…The unmethylated state of the transgenic H19 ICR did not change even at embryonic day (E) 8.5, despite the fact that allele-nonspecific methylation, probably elicited by postimplantation de novo DNA methylation activity, was observed outside of the DMR (Fig. 1G) (Matsuzaki et al, 2010). These results demonstrated that the paternally inherited transgenic H19 ICR must be recognized by the DNA methylation machinery, including DNMT3A and DNMT3L, in early embryos to acquire imprinted methylation.…”

“…Another possibility is that an epigenetic mark to designate its paternal origin might be set in this region of the H19 ICR during spermatogenesis. Importantly, this is the first example of regulatory sequences that ensure a stable methylation level of the paternal H19 ICR during preimplantation periods, because all the cis regulatory elements identified to date (such as CTCF or Sox-Oct binding motifs) function to maintain the unmethylated state of maternal H19 ICR following implantation (Matsuzaki et al, 2010;Sakaguchi et al, 2013;Schoenherr et al, 2003;Zimmerman et al, 2013).…”

“…S1D) and indistinguishable from that in blastocyst-stage embryos (Fig. S1E) (Matsuzaki et al, 2010), DNA methylation around CTCF binding site 4 of the paternally inherited H19 ICR was low in blastocysts (region II, Fig. S1F), suggesting that DNA methylation acquisition directionally extends from a region near CTCF site 1.…”

“…1A) is DNA-methylated by the DNMT3A-DNMT3L complex in prospermatogonia, the status of which is maintained on the paternal allele following fertilization (Kaneda et al, 2004;Tremblay et al, 1997), and it is thus classified as a gDMR. Whereas indispensable roles for CTCF (Matsuzaki et al, 2010;Schoenherr et al, 2003) and Sox-Oct binding motifs (Sakaguchi et al, 2013;Zimmerman et al, 2013) in maintaining maternal H19 ICR hypomethylation during postimplantation periods are well established, little is known about the underlying mechanisms that maintain paternal H19 ICR hypermethylation during preimplantation periods.…”

The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

“…1B, top). It is established that in the absence of CTCF binding sites the maternally inherited H19 ICR fragment cannot maintain its hypomethylated state in somatic cells and becomes hypermethylated after implantation, both in the endogenous locus (22) and in transgenic contexts (34). Therefore, we speculated that an unmodified DNA fragment would be hypermethylated eventually, even if it were once hypom-ethylated by the aid of H19 ICR sequences.…”