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ChIP-Seq Study Offers Clues to Immune Gene Mutation Process

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – The same enzyme that helps to mutate variable regions of immunoglobulin genes also associates with thousands of non-immunoglobulin genes in B cells, according to a study by researchers at the National Institutes of Health and Rockefeller University.

In an effort to better understand how the enzyme, called activation induced cytidine deaminase, or AID, binds to and mutates immunoglobulin genes, the team used chromatin immunoprecipitation and deep sequencing to find AID and related binding sites across the genome in B cell lymphocytes. The research, appearing online yesterday in Nature Immunology, hints that AID is drawn to stalled polymerases across the genome in B cells, but fuels mutations in immunoglobulin genes via interactions with its co-factor replication protein A, or RPA, which tends to associate with these immunoglobulin genes.

"We propose that stalled polymerase recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome," co-corresponding author Rafael Casellas, a researcher affiliated with the National Institute of Arthritis, Musculoskeletal, and Skin Diseases and the National Cancer Institute, and co-authors wrote. "Our findings provide a rationale for the oncogenic role of AID in B cell malignancy."

AID acts on single-stranded DNA during transcription, the researchers explained, causing somatic mutations in immunoglobulin genes — especially those housing immunoglobulin variable domains. Beneficial versions of the genes are subsequently selected based on exposure to antigens, they added. The enzyme also prompts a process called class switch recombination that further influences antibody gene function by swapping out one set of immunoglobulin exons with another.

Though it performs key functions in immune cells, and may have additional roles during development, the team noted, there is still much to be learned about AID targeting and regulation — particularly since off-target cytidine deaminase activity can lead to B cell lymphoma-related mutations and rearrangements.

"Despite the importance of AID in shaping the antibody response and in promoting malignancy, there is little understanding of how immunoglobulin genes are preferentially hypermutated, the extent of AID off-target activity or how AID finds its [single-stranded DNA] substrate near [transcription start sites]," they explained.

In an effort to learn more about such processes, the team did ChIP-Seq in activated mouse B cell lines using the Illumina Genome Analyzer II and antibodies targeting AID itself or targeting RPA, a single-stranded DNA binding protein and AID cofactor.

By integrating epigenetic data on dozens of epigenetic marks, gene expression data from messenger RNA sequencing experiments, information on RNA Polymerase II patterns, and more, the researchers found that AID often turns up near genes with active, open chromatin and near promoters where RNA Polymerase II is paused, including sites near thousands of non-immunoglobulin genes. RPA, on the other hand, was typically found in association with immunoglobulin genes alone.

Based on their findings so far, the team proposed that the combination of AID and RPA binding may prompt hypermutation in these immunoglobulin genes in a phosphorylation-dependent fashion. Together, they explained, this may help curb excessive off-site mutation under typical circumstances — but not in all situations.

"The large number of AID targets explains the broad genomic instability observed in primary and pre-malignant cells after sustained AID expression," the researchers explained, suggesting an improved understanding of AID binding sites may offer clues about which genes are prone to such mutation.

Moreover, they say, these and other studies might eventually help to uncover yet unappreciated functional roles for AID. "The broad recruitment of AID in the B cell genome raises the question of whether AID has additional functions beyond diversification of immunoglobulin genes," the authors argued.

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