The characteristics of CTCF binding sequences contribute to enhancer blocking activity

Area of Science:

• Genomics
• Molecular Biology
• Epigenetics

Background:

• Enhancers and promoters are well-studied gene regulatory elements.
• Insulator elements, particularly those binding CCCTC-binding factor (CTCF), are less characterized.
• Genomic context significantly influences CTCF site function and activity.

Purpose of the Study:

• To experimentally determine the enhancer-blocking activity of minimal, individual CTCF elements.
• To assess the impact of CTCF elements on gene expression during differentiation.
• To minimize the influence of genomic context by testing elements in identical locations.

Main Methods:

• Developed a novel experimental system to test individual CTCF elements.
• Assayed the ability of CTCF elements to insulate enhancers from promoters.
• Minimized genomic context effects by standardizing element location.

Main Results:

• No correlation found between CTCF element enhancer-blocking activity and evolutionary conservation.
• Resemblance to consensus core sequences or number of CTCF core motifs did not predict blocking ability.
• Strongest blockers included a previously described element upstream of the CTCF core motif.
• Uncharacterized DNaseI footprints near the core motif may influence function.

Conclusions:

• CTCF element function is not solely determined by conservation or core motifs.
• Specific sequences, including those upstream of the core motif and nearby DNaseI footprints, are critical for enhancer blocking.
• Developed a new assay for uniform and unbiased sub-classification of CTCF elements.