An imprinted GFP insertion reveals long-range epigenetic regulation in embryonic lineages

Abstract

Imprinted genes are often grouped in clusters at defined chromosomal locations. Long-range regulatory effects are implicated in the control of imprinting and these could be co-opted in the emergence of novel imprinted genes during evolution. We present a detailed analysis of a novel imprinted GFP mouse line. Tel7KI is a new insertion allele near the Ins2 locus within a cluster of imprinted genes on distal mouse Chr7. The GFP reporter becomes regulated by the host domain in two notable fashions. First, transcription of GFP is imprinted and active exclusively from the maternally inherited allele in the embryo. Second, the expressed maternal allele is subject to position effects reflecting a distinct pattern of expression. The GFP reporter acquires silencing DNA methylation marks on the paternal allele after fertilization. This imprinting is not acquired in the placenta, where GFP is active from both parental alleles, demonstrating key epigenetic differences between embryonic and extraembryonic lineages. Our analysis shows that imprinted clusters can provide environments conducive to the acquisition of imprinting upon novel inserted transcriptional units. The Tel7KI line offers new powerful avenues to explore both genetic and environmental factors implicated in the acquisition and maintenance of imprinted transcription in mammals.

An Extended Domain of Kcnq1ot1 Silencing Revealed by an Imprinted Fluorescent Reporter

“…Derivation of the Tel7KI mice (accession number MGI:3833432) was previously described (19,33). 129S1/SvImJ and C57BL/6J mice were from The Jackson Laboratory (stock numbers 002448 and 000664, respectively), CD-1 and BALB/c mice were from the University of British Columbia (UBC) Animal Care Centre.…”

“…DNA was extracted from the remaining Trizol fraction or from tissues digested with proteinase K (23). Sodium bisulfite modification and analysis of the CAG promoter from Tel7KI as well as IC1 and IC2 have previously been described (11,19,33,47). For all DNA methylation analyses by bisulfite sequencing, four independent PCRs were performed on the bisulfite-modified genomic DNA samples, and the products were individually cloned for sequencing (1 to 4 clones sequenced per PCR product).…”

“…We previously described the Tel7KI mouse line containing a green fluorescent protein (GFP) reporter inserted 2.6 kb distal to the Ins2 gene (19). In this line GFP is expressed solely from the maternal allele in postimplantation embryos while in the placenta both alleles are expressed (19).…”

“…In this line GFP is expressed solely from the maternal allele in postimplantation embryos while in the placenta both alleles are expressed (19). In the embryo, the silent paternal allele is associated with high levels of DNA methylation at the CAG promoter driving GFP (19).…”

“…In this line GFP is expressed solely from the maternal allele in postimplantation embryos while in the placenta both alleles are expressed (19). In the embryo, the silent paternal allele is associated with high levels of DNA methylation at the CAG promoter driving GFP (19). This methylation is not observed in sperm, indicating that the imprint at Tel7KI is a somatic imprint established in postimplantation embryos as a consequence of a long-range effect from one of the two imprinting centers in the region.…”