title: Complete biallelic insulation at the H19/Igf2 imprinting control region position results in fetal growth retardation and perinatal lethality.
abstract: BACKGROUND
  The H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken β-globin insulator (ChβGI)(2) are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChβGI)(2) does not become methylated in fetal male germ cells. The (ChβGI)(2) is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChβGI)(2) sequences from methylation in the male germ line.
  
  
  RESULTS
  We genetically dissected the ChβGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChβGI)(2) significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChβGI)(2) from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChβGI)(2) lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission.
  
  
  CONCLUSIONS
  In this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development.
authors.affiliation: Department of Molecular and Cellular Biology, City of Hope National Medical Center, Duarte, California, United States of America.
authors.forename: Dong-Hoon
authors.initials: DH
authors.lastname: Lee
authors.forename: Purnima
authors.initials: P
authors.lastname: Singh
authors.forename: Walter M K
authors.initials: WM
authors.lastname: Tsark
authors.forename: Piroska E
authors.initials: PE
authors.lastname: Szabó
mesh_terms: D000818:Animals; D001483:Base Sequence; D000076246:CCCTC-Binding Factor; D002645:Chickens; D019175:DNA Methylation; D005260:Female; D005313:Fetal Death / Q000235:genetics* / Q000378:metabolism; D005317:Fetal Growth Retardation / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D018392:Genomic Imprinting; D005854:Germ Cells / Q000378:metabolism; D038101:Insulator Elements; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D055544:beta-Globins / Q000235:genetics
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D055544:beta-Globins; D007335:Insulin-Like Growth Factor II