The Chicken HS4 Insulator Element Does Not Protect the <i>H19</i> ICR from Differential DNA Methylation in Yeast Artificial Chromosome Transgenic Mouse

<div><p>Mono-allelic expression at the mouse <i>IGF2/H19</i> locus is controlled by differential allelic DNA methylation of the imprinting control region (ICR). Because a randomly integrated <i>H19</i> ICR fragment, when incorporated into the genome of transgenic mice (TgM), was allele-specifically methylated in somatic, but not in germ cells, it was suggested that allele-discriminating epigenetic signature, set within or somewhere outside of the Tg <i>H19</i> ICR fragment in germ cells, was later translated into a differential DNA methylation pattern. To test if the chicken β-globin HS4 (cHS4) chromatin insulator might interfere with methylation imprinting establishment at the <i>H19</i> ICR, we inserted the <i>H19</i> ICR fragment, flanked by a set of floxed cHS4 core sequences, into a human β-globin locus YAC and generated TgM (<i>insulated ICR</i>' TgM). As controls, the cHS4 sequences were removed from one side (<i>5'HS4-deleted ICR'</i>) or both sides (<i>pseudo-WT ICR'</i>) of the insulated ICR' by <i>in vivo</i> cre-loxP recombination. The data show that while maternally inherited transgenic <i>H19</i> ICR was not methylated in <i>insulated ICR</i>' TgM, it was significantly methylated upon paternal transmission, though the level was lower than in the <i>pseudo-WT ICR</i>' control. Because this reduced level of methylation was also observed in the <i>5'HS4-deleted ICR</i>' TgM, we speculate that the phenotype is due to VEZF1-dependent demethylation activity, rather than the insulator function, borne in cHS4. Collectively, although we cannot rule out the possibility that cHS4 is incapable of blocking an allele-discriminating signal from outside of the transgene, the epigenetic signature appears to be marked intrinsically within the <i>H19</i> ICR.</p>