Research

Normally, DNA damage is considered detrimental to genomic stability. However, activation induced deaminase (AID) is a DNA cytidine deaminase mutator enzyme that induces hypermutation and DNA double-stranded breaks (DSBs) in a beneficial manner. Without AID-induced DNA lesions, B cells cannot generate mature, high-affinity or isotype-switched antibodies, resulting in compromised immunity.  

During immune responses, mature B cells diversify immunoglobulin (Ig) genes through somatic hypermutation (SHM) and class switch recombination (CSR). SHM alters antibody affinity by introducing nucleotide changes in the antigen-binding variable region of antibodies. B cells producing antibodies with improved antigen affinity are positively selected during the process of affinity maturation. CSR is a region-specific recombination reaction that replaces one antibody constant region with another, thereby altering antibody effector function while leaving the Ig variable region and its antigen-binding specificity intact.

AID initiates SHM and CSR by programmed DNA damage at Ig loci. However, AID can also induce "off-target" DNA damage, including point mutations in oncogenes such as Bcl6 and c-myc, as well as double-stranded breaks that result in oncogenic chromosome translocations such as those between c-myc and IgH (c-myc/IgH), a hallmark of Burkitt's lymphoma. The oncogenic potential of AID therefore requires strict control to maintain genomic integrity.

In addition to lymphomagenesis, recent studies have described a widening role for AID in pathology. For example, in chronic myeloid leukemia (CML), AID action mutates ABL, resulting in resistance to Imatinib and spurring pharmaceutical development of new ABL inhibitors.  Furthermore, non-lymphoid AID expression has been reported under a number of pro-inflammatory or hormonally induced conditions associated with carcinogenesis. A few examples include H. pylori infection of stomach epithelium, hepatitis infection of hepatocytes, and estrogen induction of AID in non-lymphoid tissue. Therefore, with a known role in genome destabilization and carcinogenesis, AID is a potential target for pharmaceutical intervention.

Recent studies have implicated AID and its family of deaminases (the APOBEC proteins) in removal of DNA methylation.  Deamination has been proposed to demethylate DNA by a damage and repair mechanism similar to that used in SHM. The deamination of 5-mC by AID yields thymidine, creating a T:G mismatch.  These are removed and repaired via base excision repair processes, resulting in net removal of methylation without alteration of sequence. Short or long patch repair could trigger demethylation across multiple 5-mCs, resulting in net demethylation across wider regions.

We have also recently become interested in the connection between gammaherpes virus pathogenesis and its impact on B cell development, especially with respect to Uracil DNA glycosylases (UNG) and the roles of endogenous and viral UNG (vUNG) in establishing chronic infection.

Antibodies are a central tool for epigenetic research. However, limited quantities, inconsistent quality – particularly with ChIP-seq – and the lack of modification-specific antibodies have impeded progress. Single-cell cloning technology allows B cells expressing antigen-specific antibodies to be purified directly from immunized mice via FACS. Immunoglobulin genes are amplified from single cells, cloned into expression vectors, and antibodies are produced in vitro.

Pilot project funding provided the capital required to set up the processes to clone and produce several antibodies. As a proof of principle, several antibodies have been produced from single cells and the isolation of specific antibodies for future production has been optimized. This pilot project is currently transforming into a multi-service CPRIT-funded core, supported by CPRIT grant RP190507.

Advantages of this technology over traditional hybridoma monoclonal production include: 

• Potential for limitless supply with consistent quality 
• Reconfiguration or fusion of epitope tags, fluorescent protein or HRP   
• Better suited to producing modification-specific antibodies, since screening can potentially occur prior to cloning and production

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