SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers.

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Understanding transcription factor interactions is crucial for deciphering gene regulation.
• Existing methods may lack the sensitivity or specificity to capture dynamic protein-DNA binding events.

Purpose of the Study:

• To develop and validate Split DamID (SpDamID), a novel protein complementation assay for genome-wide mapping of interacting or juxtaposed transcription factors.
• To apply SpDamID to study Notch-mediated transcriptional activation and its associated chromatin dynamics.

Main Methods:

• Split DamID (SpDamID) utilizes inactive halves of DNA adenine methyltransferase (DAM) fused to query proteins.
• Protein interaction or proximity reconstitutes DAM activity, leading to GATC adenine methylation.
• Inducible SpDamID was employed to analyze Notch signaling pathways and chromatin accessibility.

Main Results:

• SpDamID successfully mapped RBP sites bound by Notch complexes genome-wide.
• A subset of Notch complexes recruiting MAML and p300 showed altered chromatin accessibility upon Notch signaling.
• SpDamID distinguished monomeric and dimeric binding, identifying Notch dimer half-site motifs.
• Analysis revealed co-targeting of regulatory sequences by Notch and Runx1.

Conclusions:

• SpDamID is a sensitive and powerful tool for dynamic, genome-wide analysis of combinatorial protein-DNA interactions.
• The method allows for detailed investigation of transcription factor binding and its functional consequences.
• SpDamID provides new insights into Notch signaling and the co-regulation of gene expression.