Defining transcription factor nucleosome binding with Pioneer-seq.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Gene expression regulation depends on transcription factors (TFs) accessing DNA bound within nucleosomes.
• Understanding TF-nucleosome interactions is crucial for deciphering gene regulation.

Purpose of the Study:

• To develop and demonstrate a novel method, Pioneer-Seq, for comprehensively analyzing transcription factor binding to nucleosomes.
• To investigate the mechanistic requirements for pioneer factor binding to nucleosomes.

Main Methods:

• Pioneer-Seq involves creating libraries of nucleosomes with TF binding sites in all orientations.
• The method allows simultaneous examination of nucleosome positioning sequences and in vivo targeted nucleosomes (ITNs).
• TF binding to OCT4, SOX2, KLF4, and c-MYC was assessed using Pioneer-Seq.

Main Results:

• All tested TFs bind to nucleosome edges, with nucleosome sequence being the main regulatory factor.
• KLF4 demonstrated binding to a non-canonical TF binding site (TFBS) near the nucleosome dyad.
• In vivo targeted nucleosomes showed differential binding, with KLF4 and SOX2 favoring sites near the nucleosome center.

Conclusions:

• Pioneer-Seq provides a powerful tool to study TF-nucleosome interactions and TF binding mechanisms.
• Differences in TF recognition of TFBS within nucleosomes were observed, highlighting mechanistic requirements for pioneer factors.
• Nucleosome sequence and position significantly influence transcription factor accessibility and binding.