Fig. 1.

Ectopic HS5 recruits CTCF. (A) A copy of human HS5 (2.6 kbp) was floxed and introduced between HS1 and ε-globin on a human β-globin YAC (A201F4.3, inverted orientation) by homologous recombination in S. cerevisiae (20). The purified YAC DNA was used to generate lines of transgenic mice carrying either an intact transgene (HS5) or an HS5-containing transgene with a deleted CTCF site (HS5ΔCTCF). HS5 was then removed by expression of Cre recombinase, leaving a pseudo-WT control locus altered only by retention of the 34-bp Lox site. The CTCF motif within ectopic HS5 is represented by the open rectangle. (B) Adult anemic spleen chromatin was sonicated to 800- to 1000-bp fragments before performing ChIP. Conventional PCR was carried out with nested primers to amplify uniquely the endogenous and ectopic copies of HS5 (see Table S1 for primer sequences). PCR products were resolved on a 1.5% agarose gel and visualized with ethidium bromide. Ab, anti-CTCF; In, input; nA, no antibody.