Epigenetic and Phenotypic Consequences of a Truncation Disrupting the Imprinted Domain on Distal Mouse Chromosome 7

Abstract

The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.The 1-Mb imprinted domain on distal chromosome 7 (Chr 7) shares syntenic homology to the Beckwith-Wiedemann syndrome (BWS) region on human Chr 11p15.5 (69). Located less than 3 Mb from the telomere (Tel7q), this region contains two imprinting centers, IC1 and IC2. These conserved imprinting control elements are cis-acting sequences which carry opposite germ line DNA methylation marks and regulate the monoallelic expression of different flanking genes (9). In the proximal part of the Chr 7 domain, IC1 is located 2 kb upstream of the H19 promoter (Fig. 1A) (66). This sequence acquires a paternal DNA methylation imprint established during spermatogenesis and maintained throughout development (4,24,67). The methylated IC1 is required to initiate silencing of the paternal H19 allele (65), whereas the maternal IC1 controls the paternally expressed genes Igf2 and Ins2 via a methylation-sensitive insulator (6, 35). Distally, IC2 is located in intron 10 of Kcnq1 (Fig. 1A). This sequence is marked by a maternal DNA methylation imprint acquired during oogenesis (19). The unmethylated paternal IC2 is associated with the production of the Kcnq1ot1 noncoding RNA (ncRNA) and the silencing in cis of several genes in this subdomain (26). Consequently, these protein-coding genes are imprinted and expressed preferentially from the maternal Chr 7 homologue (62). These maternally expressed genes (MEGs) include transcripts expressed in the placenta and required for embryonic development, such as Ascl2 (31, 32), Cdkn1c (36), and Phlda2 (58). As in the case of IC1, IC2 appears to carry different allele-specific functions, such as promoter, enhancer, and CTCF binding insulator activities (2...

The Imprinted Phlda2 Gene Regulates Extraembryonic Energy Stores

“…Imprinted genes located at mouse distal chromosome 7 play an important role in regulating embryonic and placental growth (16,38). With regard to imprinting, this chromosomal region can be separated mechanistically into two distinct domains (7).…”

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

“…Del 7AI mice (MGI:3662901) carry a deletion of the Ascl2-Ins2 interval maintained on the outbred CD-1 background and were previously described (22). DelTel7 mice carry a chromosome 7 truncated immediately distal to Ins2 (33). The IC2KO (KvDMR1 KO) mice (14) were on the C57BL/6J strain background.…”

“…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.…”

“…Of 72 clones picked, clone 3D with an IC2KO in cis to Tel7KI was identified, expanded, and electroporated with pCAGGS-nlsCre (22) for transient Cre production to collapse the puromycin resistance marker. Note that the KIPC4-IC2KO1 ES cells contain four loxP sites inserted in the same orientation: two flanking the PGKpuro cassette and two flanking the Tel7KI insertion (33). Replicate plates were scored for GFP expression and puromycin sensitivity.…”

“…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).…”