VEZF1 Elements Mediate Protection from DNA Methylation
Figure 9
Shielding of a transgene by the multi-component HS4 barrier element.
The observed effects of insulator protein binding site mutations on the DNA methylation (this study), histone modification [14] and transcriptional status (this study and [12]) of a transgene insulated by HS4. A schematic representation is shown where HS4 harbors one CTCF (purple), one USF (green) and three VEZF1 (red) binding sites. Transcriptional activity of the reporter gene is indicated with an arrow. The histone modification status is depicted above each transgene, where high and minimal levels of H3ac and H3K4me2 are represented as ac/me and -/-, respectively. The DNA methylation status of the promoter and upstream insulators is indicated below each transgene where open and filled circles represent unmethylated and methylated bases, respectively. Shades of methylation are an approximation of the data presented in Figure 1 and Figure 2. Mutation of the CTCF site disrupts enhancer blocking activity [9] but has no effect on barrier activity of HS4. USF site mutation disrupts the recruitment of active histone modifications, resulting in transcriptional silencing. The promoter remains unmethylated in USF mutants due to the action of VEZF1. VEZF1 site mutations abrogate barrier activity despite USF-mediated recruitment of histone modifications remaining intact. VEZF1 mutants are characterized by complete promoter methylation.