Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Somatic hypermutation (SHM) generates antibody diversity by introducing mutations into immunoglobulin (Ig) genes in activated B cells.
• Diversification activators (DIVACs), acting as enhancers, are known to target SHM to specific Ig genes, but the underlying mechanism remains unclear.

Purpose of the Study:

• To investigate the mechanism by which DIVACs mediate the locus-specific targeting of somatic hypermutation.
• To determine the relationship between DIVAC activity, RNA polymerase II (Pol II) behavior, and DNA structure in mutating genes.

Main Methods:

• Experiments were conducted in chicken DT40 B cells and human Ramos Burkitt lymphoma cells.
• Analysis of RNA polymerase II (Pol II) phosphorylation, occupancy, and transcript production.
• Assessment of single-stranded DNA (ssDNA) bubble formation.
• Evaluation of histone modifications (H3K27ac) and histone variant (H3.3) levels.

Main Results:

• Highly active DIVACs increase Pol II phosphorylation and occupancy in target genes without a proportional increase in elongation-competent Pol II or full-length transcripts, indicating Pol II stalling.
• DIVAC-induced Pol II stalling shows a weak association with increased ssDNA bubble detection.
• No evidence was found for antisense transcription or altered H3K27ac or H3.3 levels mediated by DIVACs.

Conclusions:

• Pol II stalling is linked to cis-acting DIVAC elements in the context of SHM.
• DIVACs establish locus-specific targeting of SHM by creating a suitable platform for AID-mediated mutation.
• DIVACs facilitate SHM without necessarily increasing transcriptional output of the target gene.