VEZF1 Elements Mediate Protection from DNA Methylation
Figure 6
VEZF1 specifically interacts with dG–dC strings within HS4 footprints I and III and the βA-globin promoter.
(A) Gel mobility shift analysis of interactions with 32P-labelled HS4 FI, and FIII, or βA-globin promoter ‘glo wt’ oligonucleotide duplexes. Unlabelled competitor duplexes (indicated above each lane) were added at 50 fold molar excess. Adult chicken erythrocyte nuclear extract (ery) or recombinant chicken VEZF1 were used as indicated by brackets above the lanes. Arrows indicate footprint sequence-specific complexes. (B) Gel mobility supershift assays using 32P-labelled FI, FIII, FV, and glo wt oligonucleotide duplexes. Proteins were pre-incubated with anti-VEZF1 antibodies (indicated above each lane) prior to incubation with DNA. VEZF1 supershifts are evidenced by either abrogation of specific complexes (asterisks) and/or formation of low mobility ternary complexes (SS). Antibodies alone do not give rise to complexes with any of the duplexes used (data not shown). Supershift experiments shown are cropped from the full gels shown in Figure S3. (C) Sequences present in oligonucleotides used in gel mobility shift assays. dG–dC strings and their mutations are bold and underlined, respectively.