CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53

Area of Science:

• Molecular Biology
• Epigenetics
• Gene Regulation

Background:

• The CCCTC-binding factor (CTCF) is a key protein involved in gene regulation, acting as an insulator and chromatin organizer.
• CTCF's known functions primarily relate to DNA binding and its role at specific genomic loci like the p53 promoter.

Purpose of the Study:

• To identify and characterize novel functional domains within the CTCF protein.
• To investigate the role of CTCF in RNA binding and its impact on gene expression, particularly the p53 and Wrap53 transcripts.

Main Methods:

• Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation followed by deep sequencing (PAR-CLIP-seq) to identify RNA binding sites of CTCF.
• Depletion studies using cellular CTCF knockdown.
• Analysis of p53 and Wrap53 RNA levels.
• Functional assays using CTCF mutants, specifically targeting the RNA-binding region (RBR).

Main Results:

• CTCF possesses a previously unrecognized RNA-binding region (RBR), distinct from its DNA-binding domain.
• Depletion of CTCF reduced levels of both p53 mRNA and Wrap53 RNA.
• PAR-CLIP-seq confirmed genome-wide RNA binding by CTCF, including to Wrap53 RNA.
• CTCF regulates p53 expression via interaction with Wrap53 RNA.
• Cells with a mutated RBR in CTCF showed impaired p53 response to DNA damage.
• The RBR mediates RNA-dependent CTCF multimerization, potentially impacting higher-order chromatin structure.

Conclusions:

• CTCF directly binds RNA through its novel RBR, influencing the expression of key genes like p53 and Wrap53.
• The RBR is essential for CTCF's role in the p53 DNA damage response and RNA-dependent multimerization.
• These findings reveal a new layer of regulation by CTCF, involving direct RNA interaction and impacting chromatin organization.