Rasgrf1 Imprinting Is Regulated by a CTCF-Dependent Methylation-Sensitive Enhancer Blocker

Abstract

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5 of the promoter. A repeated sequence 3 of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeatmediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.Approximately 70 transcripts undergo genomic imprinting, in which expression is primarily or exclusively from one parental allele while the other allele remains silent. Accompanying allele-specific expression is allele-specific DNA methylation, which is important for imprinted expression (17). In mice, the paternal Rasgrf1 allele is exclusively expressed in neonatal brain, while the maternal allele is silent. The paternal allele is also methylated within a differentially methylated domain (DMD) located 30 kbp 5Ј of the promoter. Immediately 3Ј of the DMD is a repeated sequence element containing 40 copies of a 41-nucleotide (nt) element. DMD methylation requires the repeats. Mice lacking the repeats on the paternal allele fail to establish proper paternal specific DNA methylation during gametogenesis (11,29). Removal of the paternal repeats after fertilization but before implantation causes a loss of previously established methylation (R. Holmes, Y. Chang, and P. D. Soloway, submitted for publication). Collectively, the results show the Rasgrf1 repeats provide a positive signal for establishing and maintaining DNA methylation in mice. In other studies, sequences have been identified that regulate methylation at transgene insertion sites (4, 5, 15) and at the endogenous position of the imprinted H19/Igf2 locus (8,25,27).Paternal allele-specific expression of Rasgrf1 in neonatal brain requires DMD methylation. Mutations that cause inappropriate loss of paternal allele methylation silence paternal allele expression, while mutations that induce maternal allele methylation activate the normally silent maternal allele (11,29; Holmes et al., submitted). The correlation between DMD methylation and expression at Rasgrf1 is reminiscent of the relationship between methylation of the H19 DMD and expression of the tightly linked Igf2 locus. The H19 DMD is a methylation-sensitive enhancer-blocking element that binds CTCF when unmethylated, as on the maternal allele. This enables the enhancer-blocking activity of the DMD to prevent interaction between a downstream enhancer with the upstream Igf2 promoter, thus silencing the maternal Igf2 allele. However, when the DMD is methyla...

Functional interplay between MyoD and CTCF in regulating long-range chromatin interactions during differentiation

“…Because CTCF is known to participate in the molecular mechanisms driving allele-specific expression from the Igf2/H19 locus and other imprinted loci (Hikichi et al, 2003;Singh et al, 2012;Yoon et al, 2005), we asked whether the increase in p57 and kcnq1 expression observed after CTCF depletion resulted from a loss of imprinting. Taking advantage of responsive fibroblasts carrying single nucleotide polymorphisms in the p57/ kcnq1 locus, we have already demonstrated that the MyoDdependent induction of p57 involves upregulation of the normally active maternal allele and not the de-repression of the normally silent paternal allele (Busanello et al, 2012).…”

Genomic imprinting: employing and avoiding epigenetic processes

“…CTCF-binding sites have been identified at other imprinted genes such as Rasgrf1 and Kcnq1ot1 (Yoon et al 2005;Fitzpatrick et al 2007), but given the large number of CTCF-binding sites in the genome and the diverse proposed functions for CTCF (Barski et al 2007;Kim et al 2007;Xie et al 2007;Filippova 2008), it is unclear whether the presence of CTCF infers insulator function. There is evidence for insulator activity at the Rasgrf1 locus (Yoon et al 2005), but further experiments are required at this and other loci to determine whether they use an H19/Igf2-type model of imprinting regulation. Most importantly, additional cis-regulatory sequences, such as enhancers, must be identified to assess the suitability of an insulator model at these loci.…”

Genome-wide and parental allele-specific analysis of CTCF and cohesin DNA binding in mouse brain reveals a tissue-specific binding pattern and an association with imprinted differentially methylated regions

“…CTCF has long been associated with genomic imprinting due to its selective binding of the unmethylated maternal allele of the Igf2/H19 ICR resulting in parent-of-origin-specific expression of Igf2 and H19 in mouse and human (Bell and Felsenfeld 2000;Hark et al 2000;Kanduri et al 2000;Fedoriw et al 2004;Szabo et al 2004). CTCF has been studied at several other imprinted loci, and it binds the unmethylated allele at the gDMRs of Rasgrf1, Peg13, Kcnq1ot1 (Yoon et al 2005;Fitzpatrick et al 2007;Singh et al 2011), and Grb10 (Hikichi et al 2003;Mukhopadhyay et al 2004). CTCF-mediated regulation is postulated to be one of two major control mechanisms operating at ICRs (Lewis and Reik 2006;Kim et al 2009).…”