Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region

The imprinted expression of the mouse Igf2/H19 locus is governed by the differential methylation of the imprinting control region (ICR), which is established initially in germ cells and subsequently maintained in somatic cells, depending on its parental origin. By grafting a 2.9-kbp H19 ICR fragment into a human ␤-globin yeast artificial chromosome in transgenic mice, we previously showed that the ICR could recapitulate imprinted methylation and expression at a heterologous locus, suggesting that the H19 ICR in the ␤-globin locus contained sufficient information to maintain the methylation mark (K. Tanimoto Curiously, however, the transgenic H19 ICR was not methylated in sperm, which was distinct from that seen in the endogenous locus. Here, we reevaluated the ability of the H19 ICR to mark the parental origin using more rigid criteria. In the testis, the methylation levels of the solitary 2.9-kbp transgenic ICR fragment varied significantly between six transgenic mouse lines. However, in somatic cells, the paternally inherited ICR fragment exhibited consistently higher methylation levels at five out of six randomly integrated sites in the mouse genome. These results clearly demonstrated that the H19 ICR could acquire parent-of-origin-dependent methylation after fertilization independently of the chromosomal integration site or the prerequisite methylation acquisition in male germ cells.

Section: Discussionmentioning

confidence: 99%

A Randomly Integrated Transgenic H19 Imprinting Control Region Acquires Methylation Imprinting Independently of Its Establishment in Germ Cells

Matsuzaki

Okamura

Shimotsuma

et al. 2009

“…First, the cohesin subunits were still detectable even when micrococcal nuclease (MNase), DNase, and RNase were added to the nuclear extract before immunoprecipitation (data not shown). Second, as a control, we carried out parallel experiments with protein Ku70, which binds to some specific DNA sites adjacent to some CTCF sites (21) and can coprecipitate with CTCF (Fig. 1).…”

Section: Vol 31 2011 Cohesin-ctcf Interactions Mediated By Sa2 2175mentioning

confidence: 99%

Specific Sites in the C Terminus of CTCF Interact with the SA2 Subunit of the Cohesin Complex and Are Required for Cohesin-Dependent Insulation Activity

Xiao

Wallace

Felsenfeld

2011

175

155

Recent studies have shown that the protein CTCF, which plays an important role in insulation and in large-scale organization of chromatin within the eukaryotic nucleus, depends for both activities on recruitment of the cohesin complex. We show here that the interaction of CTCF with the cohesin complex involves direct contacts between the cohesin subunit SA2 and specific regions of the C-terminal tail of CTCF. All other cohesin components are recruited through their interaction with SA2. Expression in vivo of CTCF mutants lacking the C-terminal domain, or with mutations at sites within it required for SA2 binding, disrupts the normal expression profile of the imprinted genes IGF2-H19 and also results in a loss of insulation activity. Taken together, our results demonstrate that specific sites on the C terminus of CTCF are essential for cohesin binding and insulator function. The only direct interaction between CTCF and cohesin involves contact with SA2, which is external to the cohesin ring. This suggests that in recruiting cohesin to CTCF, SA2 could bind first and the ring could assemble subsequently.CCCTC-binding factor (CTCF) is an evolutionarily conserved zinc finger protein that has been shown to play multiple roles in the regulation of gene expression. (1,8,9,10,25,34,42). Many of its activities probably derive from the ability of CTCF to interact with and stabilize contacts between distant sites within the nucleus (33, 36). The effect of the resulting formation of loop domains in some cases can lead to activation of gene expression by bringing together distant enhancers and promoters, but in its most intensively studied role, a CTCF binding site that lies between an enhancer and a promoter can act as an insulator element to silence a gene (5, 6). The biological significance of this activity first became apparent in studies of the imprinted control region (ICR) of the Igf2-H19 imprinted locus in mouse and human (2,13,14,18), which showed that the ICR had multiple CTCF binding sites that, on the maternal allele, served to prevent a distal enhancer from activating Igf2 expression. On the paternal allele, however, the DNA of the binding sites was methylated, CTCF could not bind, and the enhancer could activate the Igf2 promoter, accounting at least in large part for allele-specific expression.As with other transcriptional regulatory proteins, CTCF does not act alone. Work over the past several years has revealed a variety of proteins that interact with it, and a number of them appear to be important or essential in assays for insulator activity or stabilization of long-range contacts (43). Among these are the chromatin-remodeling protein CHD8 (16) and suz12, a component of the PRC2 polycomb group complex that is responsible for methylation of lysine 27 on histone H3 and is associated with formation of silencing chromatin structures (24). Why these proteins are essential to CTCF function is not completely understood. However, a more obvious connection to function may be provided by the recent discovery that CTC...

“…Because some DMRs (H19, Dlk1-Dio3, Rasgrf1, and Zdbf2) acquire DNA methylation in the sperm and many others acquire DNA methylation in oocytes, and because they both are catalyzed by a common set of enzymes, Dnmt3a, -3b, and -3L (6)(7)(8)10), DMRs like the H19 ICR must harbor cis sequences to instruct their methylation only in the sperm, or their protection against de novo DNA methylation only in the eggs, although these mechanisms are not mutually exclusive. Despite intensive efforts to identify such sequences, none have ever been discovered (11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

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

Okamura

Matsuzaki

Sakaguchi

et al. 2013

cIn the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.