Research
AID function & regulation
The enzyme AID is essential for generating protective antibodies against infections and vaccination. AID functions by introducing mutations at the antibody genes to enable their functional changes. However, AID can also mutate off-target and predispose to B cell lymphoma. We use structure-function, proteomics, molecular biology, and immunological techniques in vitro and in vivo, to elucidate the mechanisms that balance these two physiological and pathological sides of AID in B cells.
License to mutate: coupling of AID to transcription elongation
We have found that AID is widely associated to chromatin. Yet, AID mutates the antibody genes and a few hundred other genes; but most of the genome is in fact spared. We determined that the ability to mutate a given gene is defined by a specific domain of AID that couples the enzyme to transcription elongation and thereby licenses it for mutagenesis.
Methot et al. Nat. Commun. 9 (1):1248, (2018).
Mechanisms regulating AID activity via subcellular localization.
We have identified mechanisms that regulate AID subcellular localization. We found that AID requires active nuclear import because cytoplasmic retention by the eEF1A translation factor prevents its diffusion into the nucleus. The balance of these mechanisms allows antibody gene diversification while limiting AID's off-target activity. We also identified drugs that inhibit the interaction between AID and eEF1A in the first pharmacological approach to increase AID function. This finding has biotechnological and clinical implications.
Methot et al. J. Exp. Med. 212, 581–596 (2015) ; Patenaude et al. Nat. Struct. Mol. Biol. 16, 517–527 (2009).
A pathway stabilizing AID protein that can be exploited to decrease its activity.
We discovered that an immature form of AID is protected from degradation by an HSP90/DNAJA1 molecular chaperoning pathway in the cytoplasm. This pathway directly impacts the efficiency of somatic hypermutation and class switching, as well as the oncogenic activity of AID. We exploited these findings to describe pharmacological approaches to reduce AID activity in vitro and in vivo; using HSP90 inhibitors or inhibitors that prevent DNAJA1 farnesylation
Montamat-Sicotte et al. Eur J Immunol 45, 2365-76 (2015) ; Orthwein et al. EMBO J 31, 679-91 (2012); Orthwein et al. J. Exp. Med. 207, 2751-65 (2010).
The interplay between AID and DNA repair in antibody responses and cancer.
We found that the C-terminal domain of AID links the DNA damage caused by AID during class switch recombination to DNA repair by non-homologous end joining, thus preventing the accumulation of DNA damage and cell death. Our results provide an explanation for an autosomal dominant immunodeficiency syndrome in patients expressing truncated forms of AID.
We have a long interest in uracil-DNA glycosylase (UNG), the enzyme that recognizes Uracils made by AID and enables class switch recombination. We found that UNG is critical for a prompt antibody response upon acute antigenic exposure. A backup pathway mediated by MSH2/6 will produce switched antibodies in the long term, but the kinetics of isotype switching by MSH2/6 are too slow for an effective immune response.
Zahn et al. Proc Natl Acad Sci U S A.111, E988-E997 (2014); Cortizas et al. Cortizas et al. J immunol. 191, 5751-63 (2013); Zahn et al. J. Immunol. 190, 5949–5960 (2013).
In collaboration with Dr Ramiro Verdun (U. of Miami), we showed that UNG protects the chromosome telomeres of B cells from AID-induced DNA damage. In the absence of UNG, MSH2/6-dependent damage leads to sudden telomere loss.
By this action, UNG protects the fitness of normal and cancer B cells expressing AID in vivo using mouse models. Our finding can explain why UNG, despite being a presumed tumor suppressor, is never lost in human B cell neoplasms that express AID. Since inhibiting UNG in AID+ B cell lymphoma cells hampers their proliferation, we propose that UNG could be an effective drug target for cancers that express AID. Inhibiting UNG would eliminate cancer cells expressing AID, which are associated with disease progression and drug resistance.
Safavi et al, NAR Cancer (2020); Cortizas et al. J Exp Med. 213, 2459-2472 (2016).
Germinal center function
Germinal centers are the major anatomical locations of AID function. Once B cells recognize cognate antigen, they express AID and can undergo isotype switching, but for somatic hypermutation to play its role in antibody affinity maturation, it requires a selection process that takes place only when B cells join the GC. The GC segregates mutation and selection in two distinct zones, "dark" and "light", with GC B cells existing in different stage in each zone.
B cells in the dark zone use AID to mutate their antibody genes; while B cells in the light zone compete for T cell help by using their antibody molecules to capture antigen and present it to the T cells. Positively selected light zone B cells face key fate decisions. They can either go back to the dark zone and keep undergoing cycles of mutation + selection to further increase the affinity of the antibody; they exit and become memory B cells, or they can differentiate into antibody-producing "plasma" cells.
We are interested in identifying molecules that are important to regulate the GC dynamics (i.e. that regulate the stage transitions and differentiation of GC B cells).
We found that the uracil-DNA glycosylase UNG protects the fitness of germinal center B cells by protecting from the genotoxic activity of AID, notably at the telomeres. UNG does the same in B cell lymphoma and leukemia that express AID. Thus, while UNG as DNA repair enzyme would normally be considered a tumor suppressor, in the context of a cancer cell that expresses AID it becomes a tumor enabler by increasing cell fitness.
Safavi et al. NAR Cancer 2020, doi: 10.1093/narcan/zcaa019
Protein arginine methyl transferases (PRMT) catalyze the post translational modification of of arginine residues in cellular proteins, thereby regulating their function. PRMTs are emerging as critical in cancer and normal biology of different tissues. Arginine methylation is abundant and dynamic in B cells, yet little is known about their role in immune responses.
We found that PRMT5 is a major regulator of B cell biology. PRMT5 is necessary for B cell development in the bone marrow, for B cell survival upon activation at the time of the first antigen encounter, and for GC formation. Specific PRMT5 deficiency in GC B cells prevents the generation of antigen specific antibodies. The effects of PRMT5 are pleiotropic, but at least in part, it regulates B cel function via transcriptional regulation and by enforcing mRNA splicing fidelity of DNA repair and other factors.
