CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• The RAG endonuclease initiates V(D)J recombination in progenitor B cells, a crucial process for adaptive immunity.
• RAG scanning, potentially aided by cohesin, locates DNA segments for recombination, but CTCF-bound elements can impede this process.
• Distal V(H) segment utilization is thought to rely on locus contraction and diffusional access to the recombination center.

Purpose of the Study:

• To investigate the role of linear RAG scanning in distal V(H) segment usage.
• To determine the impact of cohesin and CTCF on RAG scanning and V(H)-to-DJ(H) joining in G1-arrested pro-B cells.
• To explore how modulating CTCF activity affects Igh locus accessibility and recombination.

Main Methods:

• Utilized an auxin-inducible system to degrade RAD21 (a cohesin component) or CTCF in G1-arrested v-Abl pro-B cell lines.
• Assessed V(D)J recombination efficiency and chromatin interactions following protein degradation.
• Compared recombination patterns in manipulated cells with those of 'locus-contracted' primary pro-B cells.

Main Results:

• RAD21 degradation abolished V(D)J recombination and RAG scanning interactions, except for diffusion-mediated DQ52-to-J(H) joining.
• CTCF degradation suppressed CTCF-bound element interactions but significantly promoted distal V(H) segment recombination and interactions.
• The recombination patterns after CTCF degradation mimicked those observed in locus-contracted primary pro-B cells.

Conclusions:

• Cohesin-mediated RAG scanning is essential for V(D)J recombination.
• CTCF acts as a barrier to RAG scanning, preventing access to distal V(H) segments.
• Downregulating CTCF facilitates cohesin-driven RAG scanning across the Igh locus, promoting distal V(H) utilization.