Figure 6.

Binding of recombinant CTCF constructs to APP[WT] and APP[–94]. (A) DNase I footprinting of fragments extending from position –193 to +100 in the wild-type APP promoter (lanes 1 and 2) or of fragments in which the region between positions –94 and –193 had been replaced with upstream vector sequences in plasmid APP[–94] (lanes 3–7). Fragments were 5′-end-labeled at position –193 and digested with DNase I in the absence of CTCF (lanes 1, 3 and 7), with full-length recombinant CTCF (lanes 2 and 4), or with internal zinc finger deletions Zn4 (lane 5) and Zn9 (lane 6). The protected domain of full-length CTCF bound to the wild-type APP sequence (FL CTCF[WT]) is indicated by a bracket between positions –78 and –116. Position –94, representing the point of divergence between the wild-type APP sequence (WT) and APP[–94] is indicated by an arrowhead. The protected domains of full-length CTCF and N-terminal zinc finger deletions bound to the APP[–94] sequence are indicated by brackets from position –78 to –98 (FL[–94])and –83 to –98 (ΔN-Zn), respectively. (B) DNase I footprinting of fragments extending from position –193 to +100 in plasmid APP[–94] in the absence of CTCF (lane 1) or with bound full-length CTCF (lane 2). The DNase I footprinting reactions were co-electrophoresed with a dideoxy sequencing ladder obtained with the same ³²P-labeled oligonucletide used for PCR amplification (lanes 3–6). The sequence within the bracketed region is provided together with the outlines of DNase I protected domains obtained with full-length CTCF (FL[–94]) and internal deletion Zn4 (ΔN-Zn), The sequence of the APBβ binding sequence is boxed. The boundaries of the footprints are defined by the terminal nucleotides protected from DNase I digestion and their position should be considered accurate within 1 bp.