title: Targeting of >1.5 Mb of human DNA into the mouse X chromosome reveals presence of cis-acting regulators of epigenetic silencing.
abstract: Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.
authors.affiliation: Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Yang
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: McLeod
authors.forename: Allison M
authors.initials: AM
authors.lastname: Cotton
authors.forename: Charles N
authors.initials: CN
authors.lastname: de Leeuw
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Laprise
authors.forename: Kathleen G
authors.initials: KG
authors.lastname: Banks
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Simpson
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Brown
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D018899:CpG Islands; D019175:DNA Methylation; D021901:DNA, Intergenic; D044127:Epigenesis, Genetic; D005260:Female; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics; D000091402:Prohibitins; D011401:Promoter Regions, Genetic; D017475:Receptors, Nerve Growth Factor / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D019076:Transgenes; D014960:X Chromosome; D049951:X Chromosome Inactivation; D000097577:Homeobox Protein PITX2
chemical_list: D021901:DNA, Intergenic; D018398:Homeodomain Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; D009419:Nerve Tissue Proteins; D000091402:Prohibitins; D017475:Receptors, Nerve Growth Factor; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2; C484065:NGFR protein, human; C000717328:PHB protein, human